Methods for treating cancer

ABSTRACT

Disclosed herein are methods of inhibiting tumor growth or producing tumor regression in a subject by administering to the subject a therapeutically effective amount of a combination of palbociclib and RAD1901.

CROSS REFERENCE TO RELATED APPLICATION

This application is a continuation of application of U.S. patentapplication Ser. No. 15/794,861, filed Oct. 26, 2017, which is acontinuation of International Application No. PCT/US2016/030321, filedApr. 29, 2016, which claims the benefit under 35 U.S.C. § 119 of U.S.Provisional Application No. 62/154,699, filed Apr. 29, 2015, U.S.Provisional Application No. 62/155,451, filed Apr. 30, 2015, U.S.Provisional Application No. 62/158,469, filed May 7, 2015, U.S.Provisional Application No. 62/192,940, filed Jul. 15, 2015, U.S.Provisional Application No. 62/192,944, filed Jul. 15, 2015, U.S.Provisional Application No. 62/252,085, filed Nov. 6, 2015, U.S.Provisional Application No. 62/252,916, filed Nov. 9, 2015, U.S.Provisional Application No. 62/265,658, filed Dec. 10, 2015, U.S.Provisional Application No. 62/265,663, filed Dec. 10, 2015, and U.S.Provisional Application No. 62/265,696, filed Dec. 10, 2015, U.S.Provisional Application No. 62/265,774, filed Dec. 10, 2015, U.S.Provisional Application No. 62/323,572, filed Apr. 15, 2016, U.S.Provisional Application No. 62/323,576, filed Apr. 15, 2016. The entirecontents of the aforementioned applications are hereby incorporated byreference herein in their entirety, including drawings.

BACKGROUND

Breast cancer is divided into three subtypes based on expression ofthree receptors: estrogen receptor (ER), progesterone receptor (PR), andhuman epidermal growth factor receptor-2 (Her2). Overexpression of ERsis found in many breast cancer patients. ER-positive (ER+) breastcancers comprise two-thirds of all breast cancers. Other than breastcancer, estrogen and ERs are associated with, for example, ovariancancer, colon cancer, prostate cancer and endometrial cancer.

ERs can be activated by estrogen and translocate into the nucleus tobind to DNA, thereby regulating the activity of various genes. See,e.g., Marino et al., “Estrogen Signaling Multiple Pathways to ImpactGene Transcription,” Curr. Genomics 7(8): 497-508 (2006); and Heldringet al., “Estrogen Receptors: How Do They Signal and What Are TheirTargets,” Physiol. Rev. 87(3): 905-931 (2007).

Agents that inhibit estrogen production, such as aromatase inhibitors(AIs, e.g., letrozole, anastrozole and aromasin), or those that directlyblock ER activity, such as selective estrogen receptor modulators(SERMs, e.g., tamoxifen, toremifene, droloxifene, idoxifene, raloxifene,lasofoxifene, arzoxifene, miproxifene, levormeloxifene, and EM-652 (SCH57068)) and selective estrogen receptor degraders (SERDs, e.g.,fulvestrant, TAS-108 (SR16234), ZK191703, RU58668, GDC-0810 (ARN-810),GW5638/DPC974, SRN-927, ICI182782 and AZD9496), have been usedpreviously or are being developed in the treatment of ER-positive breastcancers.

SERMs (e.g., tamoxifen) and AIs are often used as a first-line adjuvantsystemic therapy for ER-positive breast cancer. Tamoxifen is commonlyused for ER-positive breast cancer. AIs suppress estrogen production inperipheral tissues by blocking the activity of aromatase, which turnsandrogen into estrogen in the body. However, AIs cannot stop the ovariesfrom making estrogen, Thus, AIs are mainly used to treat postmenopausalwomen. Furthermore, as AIs are much more effective than tamoxifen withfewer serious side effects, AIs may also be used to treat premenopausalwomen with their ovarian function suppressed. See, e.g., Francis et al.,“Adjuvant Ovarian Suppression in Premenopausal Breast Cancer,”N. Engl.J. Med, 372:436-446 (2015).

While initial treatment with these agents may be successful, manypatients eventually relapse with drug-resistant breast cancers.Mutations affecting the ER have emerged as one potential mechanism forthe development of this resistance. See, e.g., Robinson et al.,“Activating ESR1 mutations in hormone-resistant metastatic breastcancer,” Nat. Genet. 45:1446-51 (2013). Mutations in the ligand-bindingdomain (LBD) of ER are found in 21% metastatic ER-positive breast tumorsamples from patients who received at least one line of endocrinetreatment. Jeselsohn, et al., “ESR1 mutations—a mechanism for acquiredendocrine resistance in breast cancer,” Nat. Rev. Clin. Oncol.,12:573-83 (2015).

Fulvestrant is currently the only SERD approved for the treatment ofER-positive metastatic breast cancers with disease progression followingantiestrogen therapy. Despite its clinical efficacy, the utility offulvestrant has been limited by the amount of drug that can beadministered in a single injection and by reduced bioavailability.Imaging studies using 18F-fluoroestradiol positron emission tomography(FES-PET) suggest that even at the 500 mg dose level, some patients maynot have complete ER inhibition, and insufficient dosing may be a reasonfor therapeutic failure.

Another challenge associated with estrogen-directed therapies is thatthey may have undesirable effects on uterine, bone, and other tissues.The ER directs transcription of estrogen-responsive genes in a widevariety of tissues and cell types. These effects can be particularlypronounced as endogenous levels of estrogen and other ovarian hormonesdiminish during menopause. For example, tamoxifen can cause bonethinning in premenopausal women and increase the risk of endometrialcancer because it acts as a partial agonist on the endometrium. Inpostmenopausal women, AIs can cause more bone loss and more broken bonesthan tamoxifen. Patients treated with fulvestrant may also be exposed tothe risk of osteoporosis due to its mechanism of action.

Cell cycle regulators such as cyclins and cyclin-dependent kinases(CDKs) have been reported to have effects on ER expression. Lamb et al.,“Cell cycle regulators cyclin D1 and CDK4/6 have estrogenreceptor-dependent divergent functions in breast cancer migration andstem cell-like activity,” Cell Cycle 12(15): 2384-2394 (2013). SelectiveCDK4/6 inhibitors (e.g., ribociclib, abemaciclib and palbociclib) haveenabled tumor types in which CDK4/6 has a pivotal role in theG1-to-S-phase cell cycle transition to be targeted with improvedeffectiveness and fewer adverse effects to normal cells. O'Leary et al.,“Treating cancer with selective CDK4/6 inhibitors,” Nat. Rev. Clin.Oncol. (2016), published online 31 Mar. 2016(www.nature.com/nrclinonc/journal/vaop/ncurrent/full/nrclinonc.2016.26.html).The selective CDK4/6 inhibitors demonstrated the best responses whentested in combination with endocrine therapy in patients withER-positive breast cancer.

Palbociclib in combination with the aromatase inhibitor letrozole(PALoMA-1/TRIO 18 study) was approved for the treatment of hormonereceptor (HR)-positive (HR+), HER2− negative (HER2−) advanced breastcancer as initial endocrine based therapy in postmenopausal women inFebruary 2015. In February 2016, palbociclib in combination with theSERD fulvestrant (PALOMA-3 study) was approved for the treatment of ER+,HER2− advanced or metastatic breast cancer patients that had progressedon prior endocrine therapy. The FDA has granted the CDK4/6 inhibitorabemaciclib (LY2835219) a breakthrough therapy designation asmonotherapy for heavily pretreated patients with refractory HR-positiveadvanced breast cancer, based on data from a phase I study (JPBA study).Additional combinations of selective CDK4/6 inhibitors (e.g.,ribociclib, abemaciclib and palbociclib) with endocrine therapies (e.g.,AIs, SERMs and SERDs) are currently under development.

However, CDK4/6 inhibitors demonstrate toxicities that may requireintermittent therapy (O'Leary). Furthermore, there remains a need formore durable and effective ER-targeted therapies that can overcomechallenges associated with the current endocrine therapies, whileproviding additional benefits by combining with CDK4/6 inhibitors tocombat cancer in advanced stage and/or with resistance to priortreatments.

BRIEF DESCRIPTION OF DRAWINGS AND TABLES

This application contains at least one drawing executed in color. Copiesof this application with color drawing(s) will be provided by the Officeupon request and payment of the necessary fees.

FIG. 1. RAD1901-palbociclib combination showed improved tumor growthinhibition (TGI) compared to RAD1901 single agent treatment in variouspatient-derived xenograft (PDx) models regardless of ESR1 status andprior endocrine therapy. Percentage of TGI in PDx models treated withRAD1901 alone or in combination with palbociclib is shown.

FIGS. 2A-C. The combination of RAD1901 and palbociclib demonstratedtumor growth inhibition and regression in wild-type (WT) ERα MCF-7xenograft models (PR+, HER2−). (A): Tumor growth of MCF-7 xenograftmodels treated with vehicle control, palbociclib (45 mg/kg, p.o., q.d),fulvestrant (3 mg/dose, s.c., qwk), a combination of fulvestrant (3mg/dose, s.c., qwk) and palbociclib (45 mg/kg, p.o., q.d), RAD1901 (60mg/kg, p.o., q.d.), and a combination of RAD1901 (60 mg/kg, p.o., q.d.)and palbociclib (45 mg/kg, p.o., q.d); One-way ANOVA, “ns” is notsignificant, *p-value<0.05, and ***p-value<0.001; (B): Change inindividual tumor size from baseline to end of study of MCF-7 xenograftmodels treated with vehicle control, palbociclib (45 mg/kg, p.o., q.d),fulvestrant (3 mg/dose, s.c., qwk), a combination of fulvestrant (3mg/dose, s.c., qwk) and palbociclib (45 mg/kg, p.o., q.d), RAD1901 (60mg/kg, p.o., q.d.), and combinations of RAD1901 (60 mg/kg, p.o., q.d.)and palbociclib (45 mg/kg, p.o., q.d); (C): Tumor growth of MCF-7xenograft models treated with vehicle control, palbociclib (45 mg/kg,p.o., q.d), fulvestrant (3 mg/dose, s.c., qwk), a combination offulvestrant (3 mg/dose, s.c., qwk) and palbociclib (45 mg/kg, p.o.,q.d), RAD1901 (30 or 60 mg/kg, p.o., q.d.), and a combination of RAD1901(30 or 60 mg/kg, p.o., q.d.) and palbociclib (45 mg/kg, p.o., q.d).

FIGS. 3A-B. The combination of RAD1901 and palbociclib demonstratedtumor growth inhibition and regression in WT ERα PDx-11 models (PR+,Her2+, previously treated with aromatase inhibitor, fulvestrant, andchemotherapy). (A): Tumor growth of PDx-11 models treated with vehiclecontrol, fulvestrant (3 mg/dose, s.c., qwk), palbociclib (45 mg/kg,p.o., q.d), RAD1901 (60 mg/kg, p.o., q.d.), and a combination of RAD1901(60 mg/kg, p.o., q.d.) and palbociclib (45 mg/kg, p.o., q.d); (B):Change in individual tumor size from baseline to end of study in PDx-11models treated with vehicle control, fulvestrant (3 mg/dose, s.c., qwk),RAD1901 (60 mg/kg, p.o., q.d.), and a combination of RAD1901 (60 mg/kg,p.o., q.d.) and palbociclib (45 mg/kg, p.o., q.d). n=8-10/group.

FIGS. 4A-B. The combination of RAD1901 and palbociclib demonstratedtumor growth inhibition in WT ER+ PDx-2 models (PR+, Her2+, treatmentnaïve). (A): Tumor growth of PDx-2 models treated with vehicle control,RAD1901 (60 mg/kg, p.o., q.d.), fulvestrant (3 mg/dose, s.c., qwk), anda combination of RAD1901 (60 mg/kg, p.o., q.d.) and fulvestrant (3mg/dose, s.c., qwk); (B): Tumor growth of PDx-2 models treated withvehicle control, palbociclib (75 mg/kg, p.o., q.d), RAD1901 (60 mg/kg,p.o., q.d.), and a combination of RAD1901 (60 mg/kg, p.o., q.d.) andpalbociclib (75 mg/kg, p.o., q.d). n=8-10/group.

FIG. 5. Efficacy of RAD1901 sustained at least two months after RAD1901treatment ended while estradiol treatment continued in WT ERα PDx-4models (PR+, Her2+, treatment naïve).

FIGS. 6A-C. The combination of RAD1901 and palbociclib demonstratedtumor growth inhibition in mutant (Y537S) ERα PDx-5 models (PR+, Her2+,previously treated with aromatase inhibitors). (A): Tumor growth ofPDx-5 models treated with vehicle control, fulvestrant (3 mg/dose, s.c.,qwk), RAD1901 (60 mg/kg, p.o., q.d.), palbociclib (75 mg/kg, p.o., q.d),and a combination of RAD1901 (60 mg/kg, p.o., q.d.) and palbociclib (75mg/kg, p.o., q.d); (B): Change in individual tumor size from baseline today 17 for fulvestrant (3 mg/dose, s.c., qwk), palbociclib (75 mg/kg,p.o., q.d), and a combination of RAD1901 (60, 120 mg/kg, p.o., q.d.) andpalbociclib (75 mg/kg, p.o., q.d); and (C) Change in individual tumorsize from baseline to day 56 for palbociclib (75 mg/kg, p.o., q.d),RAD1901 (60, 120 mg/kg, p.o., q.d.) and a combination of RAD1901 (60mg/kg, p.o., q.d.) and palbociclib (75 mg/kg, p.o., q.d). n=8-10/group.

FIGS. 7A-B. The combination of RAD1901 and palbociclib demonstratedtumor growth inhibition in mutant (Y537S) ERα PDx-5 models (PR+, Her2+,previously treated with aromatase inhibitors). (A): Tumor growth ofPDx-5 models treated with vehicle control, fulvestrant (3 mg/dose, s.c.,qwk), RAD1901 (60 mg/kg, p.o., q.d.), palbociclib (p.o., q.d), and acombination of RAD1901 (60 mg/kg, p.o., q.d.) and palbociclib (p.o.,q.d); (B): Tumor growth of PDx-5 models treated with vehicle control,fulvestrant (3 mg/dose, s.c., qwk), RAD1901 (120 mg/kg, p.o., q.d.),palbociclib (p.o., q.d), and a combination of RAD1901 (120 mg/kg, p.o.,q.d.) and palbociclib (p.o., q.d).

FIG. 8. The combination of RAD1901 and palbociclib demonstrated tumorgrowth inhibition in mutant (Y537S) ERα PDx-5 models (PR+, Her2+,previously treated with aromatase inhibitors). Tumor growth of PDx-5models treated with vehicle control, fulvestrant (3 mg/dose, s.c., qwk),RAD1901 (60 mg/kg, p.o., q.d.), palbociclib (75 mg, p.o., q.d), acombination of fulvestrant (3 mg/dose, s.c., qwk) and palbociclib (75mg, p.o., q.d), and a combination of RAD1901 (60 mg/kg, p.o., q.d.) andpalbociclib (75 mg, p.o., q.d). n=8-10/group.

FIG. 9. Pharmacokinetic analysis of fulvestrant in nude mice. The plasmaconcentration of fulvestrant at 1 mg/dose (solid diamond), 3 mg/dose(solid circle), and 5 mg/dose (solid triangle) is shown. The nude micewere dosed subcutaneously with fulvestrant on Day 1 and the second doseon Day 8. The plasma concentration of fulvestrant was monitored at theindicated time points for up to 168 hours after the second dose.

FIG. 10. Effect of RAD1901 and fulvestrant (Faslodex) on mouse survivalin an intracranial MCF-7 tumor model.

FIGS. 11A-C. A representative image of FES-PET scan of the uterus of asubject treated with 200 and 500 mg RAD1901 p.o., q.d., and change ofthe ER engagement after the RAD1901 treatments. (A): Transversal view ofuterus CT scan before 200 mg RAD1901 treatment (a) and after (c), andtransversal view of uterus FES-PET scan before the RAD1901 treatment (b)and after (d); (B): Sagittal view of uterus CT scan before 500 mgRAD1901 treatment (top (a) panel) and after (bottom (a) panel), sagittalview of uterus FES-PET scan before the RAD1901 treatment (top (b) panel)and after (bottom (b) panel), transversal view of uterus CT scan beforethe RAD1901 treatment (top (c) panel) and after (bottom (c) panel),transversal view of uterus FES-PET scan before the RAD1901 treatment(top (d) panel) and after (bottom (d) panel); (C): % change of ERengagement after the RAD1901 treatments of Subjects 1-3 (200 mg) andSubjects 4-7 (500 mg) compared to baseline (before RAD1901 treatment).

FIGS. 12A-B. A representative image of FES-PET scan of the uterus (A)and pituitary (B) before (Baseline) and after (Post-treatment) RAD1901treatment (500 mg). (a) Lateral cross-section; (b) longitudecross-section; and (c) longitude cross-section.

FIG. 13. PR and ER expression in MCF-7 xenograft models treated withvehicle control, RAD1901, palbociclib, a combination of RAD1901 andpalbociclib, fulvestrant, and a combination of fulvestrant andpalbociclib.

FIGS. 14A-B. RAD1901 treatment resulted in complete ER degradation andinhibited ER signaling in MCF-7 cell lines (A) and T47D cell lines (B)in vitro. The ER expression was shown in both cell lines treated withRAD1901 and fulvestrant at various concentrations of 0.001 μM, 0.01 μM,0.1 μM and 1 μM, respectively. ER signaling was shown by three ER targetgenes tested: PGR, GREB1 and TFF1.

FIGS. 15A-C. RAD1901 treatment resulted in ER degradation and abrogationof ER signaling in MCF-7 xenograft models. (A): Western blot showing PRand ER expression in the MCF-7 xenograft models treated with vehiclecontrol, RAD1901 at 30 and 60 mg/kg, and fulvestrant at 3 mg/dose, 2hour or 8 hour after the last dose; (B): ER protein expression in theMCF-7 xenograft models treated with vehicle control, RAD1901 at 30 and60 mg/kg, and fulvestrant at 3 mg/dose, 2 hour after the last dose; (C):PR protein expression in the MCF-7 xenograft models treated with vehiclecontrol, RAD1901 at 30 and 60 mg/kg, and fulvestrant at 3 mg/dose, 8hour after last dose.

FIGS. 16A-C. RAD1901 treatment resulted in a rapid decrease in PR inMCF-7 xenograft models. (A): Western blot showing PR expression in MCF-7xenograft models treated with vehicle control and RAD1901 at 30, 60, and90 mg/kg, at 8 hours or 12 hours after single dose; (B): Western blotshowing PR expression in MCF-7 xenograft models treated with vehiclecontrol and RAD1901 at 30, 60, and 90 mg/kg, at 4 hours or 24 hoursafter the 7th dose; (C): Dose-dependent decrease in PR expression inMCF-7 xenograft models treated with RAD1901 at 30, 60, and 90 mg/kg.

FIGS. 17A-B. RAD1901 treatment resulted in a rapid decrease inproliferation in MCF-7 xenograft models. (A): A representativephotograph of a sectioned tumor harvested from MCF-7 xenograft modelstreated with vehicle control and RAD1901 at 90 mg/kg, 8 hours aftersingle dose and 24 hours after the 4th dose, stained for proliferationmarker Ki-67; (B): Histogram showing decrease of proliferation markerKi-67 in MCF-7 xenograft models treated with vehicle control and RAD1901at 90 mg/kg, 8 hours after single dose and 24 hours after the 4th dose.

FIG. 18. RAD1901 treatment at 30, 60, and 120 mg/kg decreased Ki67 moresignificantly than fulvestrant (1 mg/animal) in end of study tumors ofPDx-4 models four hours on the last day of a 56 day efficacy study.

FIG. 19. RAD1901 treatment at 60 and 120 mg/kg resulted in reduced ERsignaling in vivo in PDx-5 models with decreased PR expression.

FIGS. 20A-D. Effect of RAD1901 on uterine tissue in newly weaned femaleSprague-Dawley rats. (A): Uterine wet weights of rats euthanized 24hours after the final dose; (B): Epithelial height in tissue sections ofthe uterus; (C): Representative sections of Toluidine Blue O-staineduterine tissue at 400× magnification, arrows indicate uterineepithelium; (D): Total RNA extracted from uterine tissue and analyzed byquantitative RT-PCR for the level of complement C3 expression relativeto the 18S ribosomal RNA housekeeping gene.

FIG. 21. Plasma pharmacokinetic results of RAD1901 at 200, 500, 750, and1000 mg/kg after dosing on Day 7.

FIG. 22. 3ERT (I).

FIG. 23. 3ERT (II).

FIG. 24. Superimpositions of the ERα LBD-antagonist complexes summarizedin Table 10.

FIGS. 25A-B. Modeling of (A) RAD1901-1R5K; and (B) GW5-1R5K.

FIGS. 26A-B. Modeling of (A) RAD1901-1SJ0; and (B) E4D-1SJ0.

FIGS. 27A-B. Modeling of (A) RAD1901-2JFA; and (B) RAL-2JFA.

FIGS. 28A-B. Modeling of (A) RAD1901-2BJ4; and (B) OHT-2BJ4.

FIGS. 29A-B. Modeling of (A) RAD1901-2IOK; and (B) IOK-2IOK.

FIG. 30. Superimpositions of the RAD1901 conformations resulted from IFDanalysis with 1R5K and 2OUZ.

FIG. 31. Superimpositions of the RAD1901 conformations resulted from IFDanalysis with 2BJ4, and 2JFA.

FIGS. 32A-B. Superimpositions of the RAD1901 conformations resulted fromIFD analysis with 2BJ4, 2JFA and 1SJ0.

FIGS. 33A-C. IFD of RAD1901 with 2BJ4.

FIGS. 34A-C. Protein Surface Interactions of RAD1901 docked in 2BJ4 byIFD.

FIGS. 35A-C. IFD of Fulvestrant with 2BJ4.

FIGS. 36A-B. IFD of Fulvestrant and RAD1901 with 2BJ4.

FIGS. 37A-B. Superimposions of IFD of Fulvestrant and RAD1901 with 2BJ4.

FIG. 38. RAD1901 in vitro binding assay with ERα constructs of WT andLBD mutant.

FIG. 39. Location of exemplary mutations of ERα and frequencies thereof.

Table 1. RAD1901 levels in plasma, tumor and brain of mice implantedwith MCF7 cells after treated for 40 days.

Table 2. SUV for uterus, muscle, and bone for a human subject treatedwith 200 mg dose p.o., q.d., for six days.

Table 3. SUV for uterus, muscle, and bone for a human subjects (n=4)treated with 500 mg dose p.o., q.d., for six days.

Table 4. Effect of RAD1901 on BMD in ovariectomized rats. Adult femalerats underwent either sham or ovariectomy surgery before treatmentinitiation with vehicle, E2 (0.01 mg/kg) or RAD1901 (3 mg/kg) q.d. (n=20per treatment group). BMD was measured by dual emission x-rayabsorptiometry at baseline and after 4 weeks of treatment. Data areexpressed as mean±SD. *P<0.05 versus the corresponding OVX+Veh control.BMD, bone mineral density; E2, beta estradiol; OVX, ovariectomized; Veh,vehicle.

Table 5. Effect of RAD1901 on femur microarchitecture in ovariectomizedrats. Adult female rats underwent either sham or ovariectomy surgerybefore treatment initiation with vehicle, E2 (0.01 mg/kg) or RAD1901 (3mg/kg) q.d. (n=20 per treatment group). After 4 weeks, Bonemicroarchitecture was evaluated using microcomputed tomography. Data areexpressed as mean±SD. *P<0.05 versus the corresponding OVX+Veh control.ABD, apparent bone density; BV/TV, bone volume density; ConnD,connectivity density; E2, beta estradiol; OVX, ovariectomized; TbN,trabecular number; TbTh, trabecular thickness; TbSp, trabecular spacing;Veh, vehicle.

Table 6. Key baseline demographics of Phase 1 dose escalation study ofRAD1901.

Table 7. Most frequent (>10%) treatment related AEs in a Phase 1 doseescalation study of RAD1901. AEs graded as per CTCAE v4.0. Any patientwith multiple scenarios of a same preferred term was counted only onceto the most severe grade. *>10% of patients in the total active groupwho had any related TEAEs. N=number of subjects with at least onetreatment-related AE in a given category.

Table 8. Pharmacokinetic parameters in a Phase 1 dose escalation studyof RAD1901 (Day 7).

Table 8. Pharmacokinetic parameters in a Phase 1 dose escalation studyof RAD1901 (Day 7).

Table 9. Frequency of LBD mutations.

Table 10. Differences of ER-α LBD-antagonist complexes in residue posesversus 3ERT.

Table 11. Evaluation of structure overlap of ER-α LBD-antagonistcomplexes by RMSD calculations.

Table 12. Analysis of ligand binding in ER-α LBD-antagonist complexes.

Table 13. Model evaluation for RAD1901 docking.

Table 14. Induced Fit Docking Score of RAD1901 with 1R5K, 1SJ0, 2IFA,2BJ4 and 2OUZ.

DETAILED DESCRIPTION OF THE INVENTION

As set forth in the Examples section below, a combination of RAD1901 andpalbociclib (a RAD1901-palbo combination) (structures below)demonstrated greater tumor growth inhibition than RAD1901 alone inbreast cancer xenograft models, regardless of ESR1 status, PR status andprior endocrine therapy (Example I(A)). The xenograft models treated hadtumor expressing wild-type (WT) or mutant (e.g., Y537S) ERα, with orwithout PR expression, with high or low Her2 expression, and with orwithout prior endocrine therapy (e.g., tamoxifen (tam), AI,fulvestrant), chemotherapy (chemo), Her2 inhibitors (Her2i, e.g.,trastuzumab, lapatinib), bevacizumab, and/or rituximab (FIG. 1).RAD1901-palbo combinations showed greater tumor growth inhibition (TGIof 65% or higher) in xenograft models wherein RAD1901 alone achieved aTGI of 26-64%; and RAD1901-palbo combinations showed greater tumorgrowth inhibition (TGI of 26-64%) in xenograft models wherein RAD1901alone achieved a TGI of less than 25%. RAD1901-palbo combinations showedgreater tumor regression than RAD1901 alone in xenograft models that arehighly responsive to RAD1901 treatment (TGI of 65% or higher), e.g.,PDx-11 (FIGS. 3A-B)

ER WT PDx models and ER mutant PDx models may have different level ofresponsiveness to treatment with fulvestrant alone, palbociclib alone,and/or a combination of fulvestrant and palbociclib (a ful-palbocombination). However, RAD1901-palbo combinations demonstrated improvedtumor growth inhibition and/or tumor regression compared to treatmentwith RAD1901 alone or palbociclib alone, regardless of whether the PDxmodels were responsive to fulvestrant treatment and/or ful-palbocombination treatment. In other words, RAD1901-palbo combination mayinhibit tumor growth and/or produce tumor regression in fulvestrantresistant cancers.

RAD1901-palbo combination treatment demonstrated improved tumor growthinhibition and/or tumor regression compared to treatment withfulvestrant alone or with the ful-palbo combination. For example, theRAD1901-palbo combination caused more significant tumor regression inmore WT ER+ xenograft models than treatment with fulvestrant alone,RAD1901 alone, or palbociclib alone, even though these xenograft modelshave varied responsiveness to fulvestrant treatment (e.g., MCF7 cellline xenograft model responsive to fulvestrant treatment (FIGS. 2A-C);PDx-11 model responsive to fulvestrant treatment (FIGS. 3A-B); and PDx-2model least responsive to fulvestrant treatment (FIGS. 3A-B)). TheRAD1901-palbo combination also caused more significant tumor regressionin more WT ER+ MCF7 cell line xenograft models and PDx-11 models thantreatment with a ful-palbo combination (FIGS. 2A-C and 3A-B). TheRAD1901-palbo combination provided similar effects with RAD1901 at adose of 30 mg/kg or 60 mg/kg, although RAD1901 alone at 30 mg/kg was notas effective as RAD1901 alone at 60 mg/kg in inhibiting tumor growth(FIG. 2C). Said results suggest a RAD1901-palbo combination with a lowerdose of RAD1901 (e.g., 30 mg/kg) was sufficient to maximize the tumorgrowth inhibition/tumor regression effects in said xenograft models.

The RAD1901-palbo combination demonstrated tumor regression or improvedtumor growth inhibition in mutant ER+(e.g., Y537S) PDx models hardlyresponsive to fulvestrant treatment. For example, PDx-5 is an ER Y537Smutant PDx model (PR+, Her2+, prior treatment with AI) hardly responsiveto fulvestrant treatment. RAD1901-palbo combination demonstrated tumorregression in PDx-5 model, while palbociclib alone or RAD1901 alone onlyinhibited tumor growth without causing tumor regression (FIGS. 6A-C and7A-B). Furthermore, the RAD1901-palbo combination provided similareffects with RAD1901 at a dose of 60 mg/kg or 120 mg/kg (FIGS. 7A-B),suggesting a RAD1901-palbo combination with a lower dose of RAD1901(e.g., 60 mg/kg) was sufficient to maximize the tumor growthinhibition/tumor regression effects in said PDx models. TheRAD1901-palbo combination caused more significant tumor growthinhibition than RAD1901 alone, palbociclib alone, fulvestrant alone, orthe ful-palbo combination in mutant PDx-5 models (FIG. 8). Unexpectedly,the ful-palbo combination did not enhance tumor growth inhibitionsignificantly compared to treatment with palbociclib alone in PDx-5models (FIG. 8). Thus, the addition of fulvestrant did not benefit thePDx-5 models when applied in combination with palbociclib, while theadditional of RAD1901 unexpectedly did. Furthermore, the longer thetreatment, the more significant tumor growth inhibition theRAD1901-palbo combo achieved when compared to RAD1901 treatment alone orpalbociclib treatment alone (FIG. 8). Thus, RAD1901-palbo combinationsprovide powerful anti-tumor therapy for ER+ breast cancer expressing WTor mutant ER, with or without PR expression, with high or low Her2expression, and with or without resistance to fulvestrant.

The results provided herein also show that RAD1901 can be delivered tothe brain (Example II), and said delivery improved mouse survival in anintracranial tumor model expressing wild-type ERα (MCF-7 xenograftmodel, Example I(B)). As palbociclib has been reported to cross thebrain-blood barrier (O'Leary), a RAD1901-palbo combination is likely toalso be able to cross brain-blood bather and treat ER+ tumors in brain.This represents an additional advantage over the ful-palbo combinationfor treating ER+ tumors in the brain, as fulvestrant cannot cross theblood-brain barrier (Vergotel et al., “Fulvestrant, a new treatmentoption for advanced breast cancer: tolerability versus existing agents,”Ann. Oncol., 17(2):200-204 (2006)). A combination of RAD1901 with otherCDK4/6 inhibitor(s) that can cross the blood-brain barrier (e.g.,abemaciclib (O'Leary)) may also have similar therapeutic effects on ER+tumors in brain.

RAD1901 showed sustained efficacy in inhibiting tumor growth aftertreatment ended while estradiol treatment continued (e.g., PDx-4 model).Thus, a RAD1901-palbo combination is likely to benefit patients byinhibiting tumor growth after treatment ends, especially when CDK4/6inhibitors (e.g., ribociclib, abemaciclib and palbociclib) can only beadministered intermittently due to their side effects (O'Leary).

A RAD1901-palbo combination is likely to have lower side-effects thantreatment with palbociclib alone or a combination of palbociclib withother hormone therapies (e.g., AIs such as letrozole and SERDs such asfulvestrant). For example, both AIs and fulvestrant may cause bone lossin treated patients. RAD1901 is unlikely to have similar side effects.RAD1901 was found to preferentially accumulate in tumor, with a RAD1901level in tumor v. RAD1901 level in plasma (T/P ratio) of up to about 35(Example II). Standardized uptake values (SUV) for uterus, muscle andbone were calculated for human subjects treated with RAD1901 at a doseof about 200 mg up to about 500 mg q.d. (Example III(A)). Post-doseuterine signals were close to levels from “non-target tissues” (tissuesthat do not express estrogen receptor), suggesting a completeattenuation of FES-PET uptake post-RAD1901 treatment. Almost no changewas observed in pre-versus post-treatment PET scans in tissues that didnot significantly express estrogen receptor (e.g., muscles, bones)(Example IIIA). Finally, RAD1901 treatments antagonized estradiolstimulation of uterine tissues in ovariectomized (OVX) rats (ExampleIV(A)), and largely preserved bone quality of the treated subjects. Forexample, OVX rats treated with RAD1901 showed maintained BMD and femurmicroarchitecture (Example IV(A)). Thus, the RAD1901-palbo combinationmay be especially useful for patients having osteoporosis or a higherrisk of osteoporosis.

Furthermore, RAD1901 was found to degrade wild-type ERα and abrogate ERsignaling in vivo in MCF7 cell line xenograft models, and showed adose-dependent decrease in PR in these MCF7 cell line xenograft models(Example III(B)). RAD1901 decreased proliferation in MCF7 cell linexenograft models and PDx-4 models as evidenced by a decrease inproliferation marker Ki67 in tumors harvested from the treated subjects.RAD1901 also decreased ER signaling in vivo in an ER mutant PDx modelthat was hardly responsive to fulvestrant treatment (Example III(B)).

The unexpected efficacy of RAD1901-palbo combination in tumors hardlyresponsive to fulvestrant treatments and in tumors expressing mutant ERαmay be due to the unique interactions between RAD1901 and ERα.Structural models of ERα bound to RAD1901 and other ERα-bindingcompounds were analyzed to obtain information about the specific bindinginteractions (Example V). Computer modeling showed that RAD1901-ERαinteractions are not likely to be affected by mutations in the LBD ofERα, e.g., Y537X mutant wherein X was S, N, or C; D538G; and S463P,which account for about 81.7% of LBD mutations found in a recent studyof metastatic ER positive breast tumor samples from patients whoreceived at least one line of endocrine treatment (Table 9, Example V).Thus, a combination of one or more CDK4 and/or CDK6 inhibitors asdescribed herein (e.g., ribociclib, abemaciclib and palbociclib) andRAD1901 or solvates (e.g., hydrate) or salts thereof is likely to havetherapeutic effects with relatively low side effects similar toRAD1901-palbo combinations as disclosed herein. The computer modelingresulted in identification of specific residues in the C-terminalligand-binding domains of ERα that are critical to binding, informationthat can be used to develop compounds that bind and antagonize not onlywild-type ERα but also certain mutants and variants thereof, which whencombined with a CDK4/6 inhibitor (e.g., ribociclib, abemaciclib andpalbociclib) may provide strong anti-tumor therapy with relatively lowside effects similar to RAD1901-palbo combinations as disclosed herein.

Based on these results, methods are provided herein for inhibitinggrowth or producing regression of an ERα-positive tumor in a subject inneed thereof by administering to the subject a therapeutically effectiveamount of a combination of RAD1901 or solvates (e.g., hydrate) or saltsthereof and one or more CDK4 and/or CDK6 inhibitor(s) as describedherein (e.g., ribociclib, abemaciclib and palbociclib). In certainembodiments, administration of RAD1901 or solvates (e.g., hydrate) orsalts thereof has additional therapeutic benefits in addition toinhibiting tumor growth, including for example inhibiting cancer cellproliferation or inhibiting ERα activity (e.g., by inhibiting estradiolbinding or by degrading ERα). In certain embodiments, the method doesnot provide negative effects to muscles, bones, breast, and/or uterus.

In certain embodiments, RAD1901 or solvates (e.g., hydrate) or saltsthereof modulates and/or degrades ERα and mutant ERα.

In certain embodiments of the tumor growth inhibition or tumorregression methods provided herein, methods are provided for inhibitinggrowth or producing regression of an ERα-positive tumor in a subject inneed thereof by administering to the subject a therapeutically effectiveamount of a combination of one or more CDK4 and/or CDK6 inhibitor(s) asdescribed herein (e.g., ribociclib, abemaciclib and palbociclib) andRAD1901 or a solvate (e.g., hydrate) or salt thereof. In certain ofthese embodiments, the salt thereof is RAD1901 dihydrochloride havingthe structure:

CDK4 and/or CDK6 Inhibitors

In certain embodiments, the CDK4 and/or CDK6 inhibitors include, withoutlimitation, palbociclib, abemaciclib, ribociclib, AMG925, Formula IIcompounds, Formula III compounds and Formula IV compounds as disclosedbelow, solvates thereof, salts thereof, and combinations thereof.

Abemaciclib (LY2835219, 2-pyrimidinamine,N-(5-((4-ethyl-1-piperazinyl)methyl)-2-pyridinyl)-5-fluoro-4-(4-fluoro-2-methyl-1-(1-methylethyl)-1H-benzimidazol-6-yl))

Ribociclib, (LEE011,7-cyclopentyl-N,N-dimethyl-2-((5-(piperazin-1-yl)pyridin-2-yl)amino)-7H-pyrrolo[2,3-d]pyrimidine-6-carboxamide)

Formula II compounds have a structure of Formula II, includingpharmaceutically acceptable solvates (e.g., hydrates) thereof, andpharmaceutically acceptable salts thereof:

wherein:

each X is independently a heteroatom (e.g., O, S, and N); and

each R₁ is independently selected from the group consisting of hydrogen,lower alkyl, carboxy-lower alkyl, oxygen, and cycloalkyl.

Unless otherwise specified, a lower alkyl as used herein is an alkylhaving 1, 2, 3, 4, 5, or 6 carbons.

Formula III compounds have a structure of Formula III, includingpharmaceutically acceptable solvates (e.g., hydrates) thereof, andpharmaceutically acceptable salts thereof:

-   -   wherein:    -   R₂ is selected from the group consisting of hydrogen, halogen        atoms, NH₂, NHR₂, NHCOR₂, NO₂, CN, CH₂NH₂ CH₂NHR₂, phenyl and        heteroaromatic groups, wherein the phenyl or heteroaromatic        group is optionally substituted with a further substitution        selected from the group consisting of lower alkyl, carboxy-lower        alkyl, oxygen, and cycloalkyl groups;    -   Ar is phenyl or heteroaromatic group, wherein the phenyl or        heteroaromatic group is optionally substituted with a further        substitution selected from the group consisting of lower alkyl,        carboxy-lower alkyl (—(C═O)-lower alkyl), oxygen (═O), or        cycloalkyl groups; and    -   n is 0, 1, 2 or 3.

Formula IV compounds have a structure of Formula IV as disclosed inEP1295878B1, which is herein incorporated by reference, furtherincluding pharmaceutically acceptable solvates (e.g., hydrates) thereof,and pharmaceutically acceptable salts thereof:

wherein:

Ar₂ is

and Ar is or

Ar₂ is idem and Ar is

Combination Therapy(1) Combination of RAD1901 or Solvates (e.g., Hydrate) or Salts Thereofand One or More CDK4 and/or CDK6 Inhibitor(s)

Both RAD1901 or solvates (e.g., hydrate) or salts thereof and the CDK4and/or CDK6 inhibitor(s), when administered alone to a subject, have atherapeutic effect on one or more cancers or tumors (Examples I(A) andI(B)). It was surprisingly discovered that when administered incombination to a subject, RAD1901 or solvates (e.g., hydrate) or saltsthereof and the CDK4 and/or CDK6 inhibitor(s) have a significantlyimproved effect on the cancers/tumors (Examples I(A) and I(B)).

“Inhibiting growth” of an ERα-positive tumor as used herein may refer toslowing the rate of tumor growth, or halting tumor growth entirely.

“Tumor regression” or “regression” of an ERα-positive tumor as usedherein may refer to reducing the maximum size of a tumor. In certainembodiments, administration of a combination of one or more CDK4 and/orCDK6 inhibitor(s) as described herein (e.g., ribociclib, abemaciclib andpalbociclib) and RAD1901 or solvates (e.g., hydrate) or salts thereofmay result in a decrease in tumor size versus baseline (i.e., size priorto initiation of treatment), or even eradication or partial eradicationof a tumor. Accordingly, in certain embodiments the methods of tumorregression provided herein may be alternatively characterized as methodsof reducing tumor size versus baseline.

“Tumor” as used herein is a malignant tumor, and is used interchangeablywith “cancer.”

Tumor growth inhibition or regression may be localized to a single tumoror to a set of tumors within a specific tissue or organ, or may besystemic (i.e., affecting tumors in all tissues or organs).

As RAD1901 is known to preferentially bind ERα versus estrogen receptorbeta (ERβ), unless specified otherwise, estrogen receptor, estrogenreceptor alpha, ERα, ER, wild-type ERα, and ESR1 are usedinterchangeably herein. “Estrogen receptor alpha” or “ERα” as usedherein refers to a polypeptide comprising, consisting of, or consistingessentially of the wild-type ERα amino acid sequence, which is encodedby the gene ESR1. A tumor that is “positive for estrogen receptoralpha,” “ERα-positive,” “ER+,” or “ERα+” as used herein refers to atumor in which one or more cells express at least one isoform of ERα. Incertain embodiments, these cells overexpress ERα. In certainembodiments, the patient has one or more cells within the tumorexpressing one or more forms of ERβ. In certain embodiments, theERα-positive tumor and/or cancer is associated with breast, uterine,ovarian, or pituitary cancer. In certain of these embodiments, thepatient has a tumor located in breast, uterine, ovarian, or pituitarytissue. In those embodiments where the patient has a tumor located inthe breast, the tumor may be associated with luminal breast cancer thatmay or may not be positive for HER2, and for HER2+ tumors, the tumorsmay express high or low HER2 (e.g., FIG. 1). In other embodiments, thepatient has a tumor located in another tissue or organ (e.g., bone,muscle, brain), but is nonetheless associated with breast, uterine,ovarian, or pituitary cancer (e.g., tumors derived from migration ormetastasis of breast, uterine, ovarian, or pituitary cancer).Accordingly, in certain embodiments of the tumor growth inhibition ortumor regression methods provided herein, the tumor being targeted is ametastatic tumor and/or the tumor has an overexpression of ER in otherorgans (e.g., bones and/or muscles). In certain embodiments, the tumorbeing targeted is a brain tumor and/or cancer. In certain embodiments,the tumor being targeted is more sensitive to a treatment of RAD1901 anda CDK4 and/or CDK 6 inhibitor as disclosed herein than treatment withanother SERD (e.g., fulvestrant, TAS-108 (SR16234), ZK191703, RU58668,GDC-0810 (ARN-810), GW5638/DPC974, SRN-927, ICI182782 and AZD9496), Her2inhibitors (e.g., trastuzumab, lapatinib, ado-trastuzumab emtansine,and/or pertuzumab), chemo therapy (e.g., abraxane, adriamycin,carboplatin, cytoxan, daunorubicin, doxil, ellence, fluorouracil,gemzar, helaven, lxempra, methotrexate, mitomycin, micoxantrone,navelbine, taxol, taxotere, thiotepa, vincristine, and xeloda),aromatase inhibitor (e.g., anastrozole, exemestane, and letrozole),selective estrogen receptor modulators (e.g., tamoxifen, raloxifene,lasofoxifene, and/or toremifene), angiogenesis inhibitor (e.g.,bevacizumab), and/or rituximab.

In certain embodiments of the tumor growth inhibition or tumorregression methods provided herein, the methods further comprise a stepof determining whether a patient has a tumor expressing ERα prior toadministering a combination of one or more CDK4 and/or CDK6 inhibitor(s)as described herein (e.g., ribociclib, abemaciclib and palbociclib) andRAD1901 or solvates (e.g., hydrate) or salts thereof. In certainembodiments of the tumor growth inhibition or tumor regression methodsprovided herein, the methods further comprise a step of determiningwhether the patient has a tumor expressing mutant ERα prior toadministering a combination of one or more CDK4 and/or CDK6 inhibitor(s)as described herein (e.g., ribociclib, abemaciclib and palbociclib) andRAD1901 or solvates (e.g., hydrate) or salts thereof. In certainembodiments of the tumor growth inhibition or tumor regression methodsprovided herein, the methods further comprise a step of determiningwhether a patient has a tumor expressing ERα that is responsive ornon-responsive to fulvestrant treatment prior to administering acombination of one or more CDK4 and/or CDK6 inhibitor(s) as describedherein (e.g., ribociclib, abemaciclib and palbociclib) and RAD1901 orsolvates (e.g., hydrate) or salts thereof. These determinations may bemade using any method of expression detection known in the art, and maybe performed in vitro using a tumor or tissue sample removed from thesubject.

In addition to demonstrating the ability of RAD1901 to inhibit tumorgrowth in tumors expressing wild-type ERα, the results provided hereinshow that RAD1901 exhibited the unexpected ability to inhibit the growthof tumors expressing a mutant form of ERα, namely Y537S ERα (ExampleI(A)). Computer modeling evaluations of examples of ERα mutations showedthat none of these mutations were expected to impact the LBD orspecifically hinder RAD1901 binding (Example V(A)), e.g., ERα having oneor more mutants selected from the group consisting of ERα with Y537Xmutant wherein X is S, N, or C, ERα with D538G mutant, and ERα withS463P mutant. Based on these results, methods are provided herein forinhibiting growth or producing regression of a tumor that is positivefor ERα having one or more mutants within the ligand-binding domain(LBD), selected from the group consisting of Y537X₁ wherein X₁ is S, N,or C, D538G, L536X₂ wherein X₂ is R or Q, P535H, V534E, S463P, V392I,E380Q, especially Y537S ERα, in a subject with cancer by administeringto the subject a therapeutically effective amount of a combination ofone or more CDK4 and/or CDK6 inhibitor(s) as described herein (e.g.,ribociclib, abemaciclib and palbociclib) and RAD1901 or solvates (e.g.,hydrate) or salts thereof. In certain embodiments, RAD1901 or solvates(e.g., hydrate) or salts thereof. “Mutant ERα” as used herein refers toERα comprising one or more substitutions or deletions, and variantsthereof comprising, consisting of, or consisting essentially of an aminoacid sequence with at least 80%, at least 85%, at least 90%, at least95%, at least 97%, at least 98%, at least 99%, or at least 99.5%identity to the amino acid sequence of ERα.

In addition to inhibiting breast cancer tumor growth in an animalxenograft model, the results disclosed herein show that RAD1901 exhibitssignificant accumulation within tumor cells, and is capable ofpenetrating the blood-brain barrier (Example II). The ability topenetrate the blood-brain barrier was confirmed by showing that RAD1901administration significantly prolonged survival in a brain metastasisxenograft model (Example I(B)). Accordingly, in certain embodiments ofthe tumor growth inhibition or tumor regression methods provided herein,the ERα-positive tumor being targeted is located in the brain orelsewhere in the central nervous system. In certain of theseembodiments, the ERα-positive tumor is primarily associated with braincancer. In other embodiments, the ERα-positive tumor is a metastatictumor that is primarily associated with another type of cancer, such asbreast, uterine, ovarian, or pituitary cancer, or a tumor that hasmigrated from another tissue or organ. In certain of these embodiments,the tumor is a brain metastases, such as breast cancer brain metastases(BCBM). In certain embodiments of the methods disclosed herein, RAD1901or solvates (e.g., hydrate) or salts thereof accumulate in one or morecells within a target tumor.

In certain embodiments of the methods disclosed herein, RAD1901 orsolvates (e.g., hydrate) or salts thereof preferably accumulate in tumorat a T/P (RAD1901 concentration in tumor/RAD1901 concentration inplasma) ratio of about 15 or higher, about 18 or higher, about 19 orhigher, about 20 or higher, about 25 or higher, about 28 or higher,about 30 or higher, about 33 or higher, about 35 or higher, or about 40or higher.

The results provided herein show that RAD1901 administration protectsagainst bone loss in ovariectomized rats (Example IV(A)). Accordingly,in certain embodiments of the tumor growth inhibition or tumorregression methods provided herein, administration of a combination ofone or more CDK4 and/or CDK6 inhibitor(s) as described herein (e.g.,ribociclib, abemaciclib and palbociclib) and RAD1901 or solvates (e.g.,hydrate) or salts thereof does not have undesirable effects on bone,including for example undesirable effects on bone volume density, bonesurface density, bone mineral density, trabecular number, trabecularthickness, trabecular spacing, connectivity density, and/or apparentbone density of the treated subject. As tamoxifen may be associated withbone loss in premenopausal women, and fulvestrant may impair the bonestructures due to its mechanism of action, a combination of one or moreCDK4 and/or CDK6 inhibitor(s) as described herein (e.g., ribociclib,abemaciclib and palbociclib) and RAD1901 or solvates (e.g., hydrate) orsalts thereof can be particularly useful for premenopausal women, tumorsresistant to tamoxifen or antiestrogen therapy, and patients havingosteoporosis and/or high risk of osteoporosis.

The results provided herein show that RAD1901 antagonized estradiolstimulation of uterine tissues in ovariectomized rats (Example IV(A)).Furthermore, in human subjects treated with RAD1901 at a dosage of 200mg or up to 500 mg q.d., standardized uptake value (SUV) for uterus,muscle, and bone tissues that did not significantly express ER showedhardly any changes in signals pre- and post-treatment (Example III(A)).Accordingly, in certain embodiments, such administration also does notresult in undesirable effects on other tissues, including for exampleuterine, muscle, or breast tissue.

RAD1901 or solvates (e.g., hydrate) or salts thereof and the CDK4 and/orCDK6 inhibitor (e.g., ribociclib, abemaciclib and palbociclib) disclosedherein are administered in combination to a subject in need. The phrase“in combination” means RAD1901 or solvates (e.g., hydrate) or saltsthereof may be administered before, during, or after the administrationof the CDK4 and/or CDK6 inhibitor. For example, RAD1901 or solvates(e.g., hydrate) or salts thereof and the CDK4 and/or CDK6 inhibitor(e.g., ribociclib, abemaciclib and palbociclib) disclosed herein can beadministered in about one week apart, about 6 days apart, about 5 daysapart, about 4 days apart, about 3 days apart, about 2 days apart, about24 hours apart, about 23 hours apart, about 22 hours apart, about 21hours apart, about 20 hours apart, about 19 hours apart, about 18 hoursapart, about 17 hours apart, about 16 hours apart, about 15 hours apart,about 14 hours apart, about 13 hours apart, about 12 hours apart, about11 hours apart, about 10 hours apart, about 9 hours apart, about 8 hoursapart, about 7 hours apart, about 6 hours apart, about 5 hours apart,about 4 hours apart, about 3 hours apart, about 2 hours apart, about 1hour apart, about 55 minutes apart, about 50 minutes apart, about 45minutes apart, about 40 minutes apart, about 35 minutes apart, about 30minutes apart, about 25 minutes apart, about 20 minutes apart, about 15minutes apart, about 10 minutes apart, or about 5 minutes apart. Inother embodiments RAD1901 or solvates (e.g., hydrate) or salts thereofand the CDK4 and/or CDK6 inhibitor (e.g., ribociclib, abemaciclib andpalbociclib) disclosed herein are administered to the subjectsimultaneously or substantially simultaneously. In certain of theseembodiments, RAD1901 or solvates (e.g., hydrate) or salts thereof andthe CDK4 and/or CDK6 inhibitor (e.g., ribociclib, abemaciclib andpalbociclib) disclosed herein may be administered as part of a singleformulation.

In some embodiments, the combination of RAD1901 or solvates (e.g.,hydrate) or salts thereof and a single CDK4 and/or CDK6 inhibitor isadministered to a subject. In other embodiments, the combination ofRAD1901 or solvates (e.g., hydrate) or salts thereof and more than oneCDK4 and/or CDK6 inhibitor is administered to a subject. For example,RAD1901 or solvates (e.g., hydrate) or salts thereof can be combinedwith two or more CDK4 and/or CDK6 inhibitors for treatingcancers/tumors.

(2) Dosage

A therapeutically effective amount of a combination of one or more CDK4and/or CDK6 inhibitor(s) as described herein (e.g., ribociclib,abemaciclib and palbociclib) and RAD1901 or solvates (e.g., hydrate) orsalts thereof for use in the methods disclosed herein is an amount that,when administered over a particular time interval, results inachievement of one or more therapeutic benchmarks (e.g., slowing orhalting of tumor growth, resulting in tumor regression, cessation ofsymptoms, etc.). The combination for use in the presently disclosedmethods may be administered to a subject one time or multiple times. Inthose embodiments wherein the compounds are administered multiple times,they may be administered at a set interval, e.g., daily, every otherday, weekly, or monthly. Alternatively, they can be administered at anirregular interval, for example on an as-needed basis based on symptoms,patient health, and the like. A therapeutically effective amount of thecombination may be administered q.d. for one day, at least 2 days, atleast 3 days, at least 4 days, at least 5 days, at least 6 days, atleast 7 days, at least 10 days, or at least 15 days. Optionally, thestatus of the cancer or the regression of the tumor is monitored duringor after the treatment, for example, by a FES-PET scan of the subject.The dosage of the combination administered to the subject can beincreased or decreased depending on the status of the cancer or theregression of the tumor detected.

Ideally, the therapeutically effective amount does not exceed themaximum tolerated dosage at which 50% or more of treated subjectsexperience nausea or other toxicity reactions that prevent further drugadministrations. A therapeutically effective amount may vary for asubject depending on a variety of factors, including variety and extentof the symptoms, sex, age, body weight, or general health of thesubject, administration mode and salt or solvate type, variation insusceptibility to the drug, the specific type of the disease, and thelike.

Examples of therapeutically effective amounts of a RAD1901 or solvates(e.g., hydrate) or salts thereof for use in the methods disclosed hereininclude, without limitation, about 150 to about 1,500 mg, about 200 toabout 1,500 mg, about 250 to about 1,500 mg, or about 300 to about 1,500mg dosage q.d. for subjects having resistant ER-driven tumors orcancers; about 150 to about 1,500 mg, about 200 to about 1,000 mg orabout 250 to about 1,000 mg or about 300 to about 1,000 mg dosage q.d.for subjects having both wild-type ER driven tumors and/or cancers andresistant tumors and/or cancers; and about 300 to about 500 mg, about300 to about 550 mg, about 300 to about 600 mg, about 250 to about 500mg, about 250 to about 550 mg, about 250 to about 600 mg, about 200 toabout 500 mg, about 200 to about 550 mg, about 200 to about 600 mg,about 150 to about 500 mg, about 150 to about 550 mg, or about 150 toabout 600 mg q.d. dosage for subjects having majorly wild-type ER driventumors and/or cancers. In certain embodiments, the dosage of a compoundof Formula I (e.g., RAD1901) or a salt or solvate thereof for use in thepresently disclosed methods general for an adult subject may beapproximately 200 mg, 400 mg, 30 mg to 2,000 mg, 100 mg to 1,500 mg, or150 mg to 1,500 mg p.o., q.d. This daily dosage may be achieved via asingle administration or multiple administrations.

A therapeutically effective amount or dosage of a CDK4 and/or CDK6inhibitor as described herein (e.g., ribociclib, abemaciclib andpalbociclib) depends on its particular type. In general, the dailydosage of a CDK4 and/or CDK6 inhibitor as described herein (e.g.,ribociclib, abemaciclib and palbociclib) ranges from about 1 mg to about1,500 mg, from about 1 mg to about 1,200 mg, from about 1 mg to about1,000 mg, from about 1 mg to about 800 mg, from about 1 mg to about 600mg, from about 1 mg to about 500 mg, from about 1 mg to about 200 mg,from about 1 mg to about 100 mg, from about 1 mg to about 50 mg, fromabout 1 mg to about 30 mg, from about 1 mg to about 20 mg, from about 1mg to about 10 mg, from about 1 mg to about 5 mg, from about 50 mg toabout 1,500 mg, from about 100 mg to about 1,200 mg, from about 150 mgto about 1,000 mg, from about 200 mg to about 800 mg, from about 300 mgto about 600 mg, from about 350 mg to about 500 mg.

Abemaciclib

Dosing of RAD1901 with abemaciclib can be accomplished with RAD1901 at100, 200, 300, 400, 500, 600, 700, 800, 900 or 1,000 mg per day. Inparticular, 200 mg, 400 mg, 500 mg, 600 mg, 800 mg and 1,000 mg per dayare noted. Under certain circumstances a BID dosing schedule ispreferred. The surprisingly long half-life of RAD1901 in humans after POdosing make this option particularly viable. Accordingly, the drug maybe administered as 200 mg bid (400 mg total daily), 250 mg bid (500 mgtotal daily), 300 mg bid (600 mg total daily), 400 mg bid (800 mg daily)or 500 mg bid (1,000 mg total daily). Preferably the dosing is oral. Thedose of abemaciclib may be 50 mg to 500 mg daily, or 150 mg to 450 mgdaily and the dosing can be daily in 28 day cycles or less than 28 daysper 28 day cycles such as 21 days per 28 day cycle or 14 days per 28 daycycle or 7 days per 28 day cycles. In some embodiments, the abemaciclibis dosed once daily or preferably on a bid schedule where dosing isoral. In the case of bid dosing, the doses can be separated by 4 hours,8 hours or 12 hours. In certain embodiments, the abemaciclib is dosed at150 mg bid via oral where the doses are recommended to be spaced out by12 hours.

As has been discovered and explained herein, there appears to be aremarkable synergy between RAD1901 and cdk 4/6 inhibitors and therefore,a dose reduction of RAD 1901 and/or abemaciclib from the usualrecommended or approved dose is contemplated and described herein. Forexample, RAD 1901 may be recommended for monotherapy treatment at dosesof 100, 200, 300, 400, 500, 600, 700, 800, 900 or 1,000 mg or morespecifically at 200 mg, 400 mg, 500 mg, 600 mg, 800 mg and 1,000 mg perday. In combination, a reduction of the specified dose by a givenfraction means that doses of 25% to 75% less than the usual dose arepossible. BY way of non-limiting example, a recommended dose of RAD1901of 400 mg per day may be reduced to between a final dose of 100 mg and300 mg per day, or 100 mg per day, 200 mg per day or 300 mg per day. Ifthe RAD1901 dose is reduced as described, the same percent reduction isgenerally applied whether the dosing is bid or once daily. For example,a 400 mg bid dose reduced by 50% would be administered on a 200 mg bidschedule. In some exceptions, a reduction of a daily recommended biddose may be sufficient to allow for the total daily dose to beadministered as a once daily dose. For example, a normal bid dose of 300mg that is given in combination with abemaciclib may be reduced by 50%.Accordingly, the dose may be given as 150 mg bid or 300 mg once daily.

Similarly, the normal recommended dose of abemaciclib may be reducedwhen used in combination with RAD1901. The dose of abemaciclib may bereduced and combined with the normal recommended monotherapy dose ofRAD1901 or a reduced RAD1901 dose wherein the reduced dose is 25% to 75%less than the normal recommended dose as exemplified immediately above.For example, a recommended dose of abemaciclib of 150 mg bid might begiven as a bid dose of 25% to 75% less than the 150 mg bid dose. Forexample, 150 mg bid of abemaciclib may be reduced to a bid dose of 37.5mg to 112.5 mg (total daily dose of 75 mg to 225 mg). Alternatively, itmay be desirable to reduce the frequency of abemaciclib from arecommended 28 day on cycle to some amount less. For example, the dosingfrequency can be reduced to 22 days to 27 days out of a 28 day cycle orto 21 days out of a 28 day cycle, or the dosing frequency may be reducedto 15 days to 20 days out of a 28 day cycle or to 14 days out of a 28day cycle, or the dosing frequency may be reduced to 8 days to 13 daysout of a 28 day cycle or to just 7 days out of a 28 day cycle. The daysdosed may be consecutive or combined as needed under the circumstance.In one embodiment, the total dose over a dosing interval is reduced by25% to 75% of the recommended dose and that reduction may come as aresult of less frequent dosing, reduced dosage or a combination thereof.For example, a recommended dosing cycle of 28 days of abemaciclib at adose of 150 mg bid (300 mg total daily) results in a total dose over 28days of 8,400 mg (28 days times 300 mg total per day). This amount canbe reduced to between from 2,100 mg per 28 day to 6,300 mg per 28 day.

Ribociclib

Dosing of RAD1901 with ribociclib can be accomplished with RAD1901 at100, 200, 300, 400, 500, 600, 700, 800, 900 or 1,000 mg per day. Inparticular, 200 mg, 400 mg, 500 mg, 600 mg, 800 mg and 1,000 mg per dayare noted. Under certain circumstances a BID dosing schedule preferred.The surprisingly long half-life of RAD1901 in humans after PO dosingmake this option particularly viable. Accordingly, the drug may beadministered as 200 mg bid (400 mg total daily), 250 mg bid (500 mgtotal daily), 300 mg bid (600 mg total daily), 400 mg bid (800 mg daily)or 500 mg bid (1,000 mg total daily). Preferably the dosing is oral. Thedose of ribociclib may be 200 mg to 1,000 mg daily, or 250 mg to 750 mgdaily and the dosing can be daily in 28 day cycles or less than 28 daysper 28 day cycles such as 21 days per 28 day cycle or 14 days per 28 daycycle or 7 days per 28 day cycles. In some embodiments, the ribociclibis dosed once daily where dosing is oral. In certain embodiments, thedose of ribociclib to be used in combination with RAD1901 is 600 mg oncedaily and the dosing interval is 21 days out of a 28 day cycle.

As has been discovered and explained herein, there appears to be aremarkable synergy between RAD1901 and cdk4/6 inhibitors and therefore,a dose reduction of RAD 1901 and/or ribociclib from the usualrecommended or approved dose is contemplated and described herein. Forexample, RAD 1901 may be recommended for monotherapy treatment at dosesof 100, 200, 300, 400, 500, 600, 700, 800, 900 or 1,000 mg or morespecifically at 200 mg, 400 mg, 500 mg, 600 mg, 800 mg and 1,000 mg perday. In combination, a reduction of the specified dose by a givenfraction means that doses of 25% to 75% less than the usual dose arepossible. BY way of non-limiting example, a recommended dose of RAD1901of 400 mg per day may be reduced to between a final dose of 100 mg and300 mg per day, or 100 mg per day, 200 mg per day or 300 mg per day. Ifthe RAD1901 dose is reduced as described, the same percent reduction isgenerally applied whether the dosing is bid or once daily. For example,a 400 mg bid dose reduced by 50% would be administered on a 200 mg bidschedule. In some exceptions, a reduction of a daily recommended biddose may be sufficient to allow for the total daily dose to beadministered as a once daily dose. For example, a normal bid dose of 300mg that is given in combination with ribociclib may be reduced by 50%.Accordingly, the dose may be given as 150 mg bid or 300 mg once daily.

Similarly, the normal recommended dose of ribociclib may be reduced whenused in combination with RAD1901. The dose of ribociclib may be reducedand combined with the normal recommended monotherapy dose of RAD1901 ora reduced RAD1901 dose wherein the reduced dose is 25% to 75% less thanthe normal recommended dose as exemplified immediately above. Forexample, a recommended dose of ribociclib of 600 mg qd might be given asa qd dose of 25% to 75% less than the 600 mg dose. For example, 600 mgof a recommended ribociclib dose may be reduced to a dose of between 150mg to 450 mg. Alternatively, it may be desirable to reduce the frequencyof ribociclib from a recommended 21 day out of 28 day on cycle to someamount less. For example, the dosing frequency can be reduced to 15 to20 days to out of a 28 day cycle or to 14 days out of a 28 day cycle, orthe dosing frequency may be reduced to 8 days to 13 days out of a 28 daycycle or to 7 days out of a 28 day cycle. The days dosed may beconsecutive or combined as needed under the circumstance. In oneembodiment, the total dose over a dosing interval is reduced by 25% to75% of the recommended dose and that reduction may come as a result ofless frequent dosing, reduced dosage or a combination thereof. Forexample, a recommended dosing cycle of 28 days of ribociclib (21 days ata dose of 600 mg qd) results in a total dose over 28 days of 12,600 mg(21 dosing days times 600 mg total per day). This amount can be reducedto between from 3,150 mg per 28 day cycle to 9,450 mg per 28 day cycle.

Palbociclib

Dosing of RAD1901 with palbociclib can be accomplished with RAD1901 at100, 200, 300, 400, 500, 600, 700, 800, 900 or 1,000 mg per day. Inparticular, 200 mg, 400 mg, 500 mg, 600 mg, 800 mg and 1,000 mg per dayare noted. Under certain circumstances a BID dosing schedule preferred.The surprisingly long half-life of RAD1901 in humans after PO dosingmake this option particularly viable. Accordingly, the drug may beadministered as 200 mg bid (400 mg total daily), 250 mg bid (500 mgtotal daily), 300 mg bid (600 mg total daily), 400 mg bid (800 mg daily)or 500 mg bid (1,000 mg total daily). Preferably the dosing is oral. Thedose of palbociclib may be 25 mg to 250 mg daily, or 50 mg to 125 mgdaily or from 75 mg to 125 mg daily or 75 mg daily or 100 mg daily or125 mg daily. The dosing can be daily in 28 day cycles or less than 28days per 28 day cycles such as 21 days per 28 day cycle or 14 days per28 day cycle or 7 days per 28 day cycles. In some embodiments, thepalbociclib is dosed once daily where dosing is oral. In certainembodiments, the dose of palbociclib to be used in combination withRAD1901 is 125 mg once daily and the dosing interval is 21 days out of a28 day cycle, or 100 mg once daily and the dosing interval is 21 daysout of a 28 day cycle or 75 mg once daily and the dosing interval is 21days out of a 28 day cycle.

As has been discovered and explained herein, there appears to be aremarkable synergy between RAD1901 and palbociclib and therefore, a dosereduction of RAD 1901 and/or palbociclib from the usual recommended orapproved dose is contemplated and described herein. For example, RAD1901 may be recommended for monotherapy treatment at doses of 100, 200,300, 400, 500, 600, 700, 800, 900 or 1,000 mg or more specifically at200 mg, 400 mg, 500 mg, 600 mg, 800 mg and 1,000 mg per day. Incombination, a reduction of the specified dose by a given fraction meansthat doses of 25% to 75% less than the usual dose are possible. BY wayof non-limiting example, a recommended dose of RAD1901 of 400 mg per daymay be reduced to between a final dose of 100 mg and 300 mg per day, or100 mg per day, 200 mg per day or 300 mg per day. If the RAD1901 dose isreduced as described, the same percent reduction is generally appliedwhether the dosing is bid or once daily. For example, a 400 mg bid dosereduced by 50% would be administered on a 200 mg bid schedule. In someexceptions, a reduction of a daily recommended bid dose may besufficient to allow for the total daily dose to be administered as aonce daily dose. For example, a normal bid dose of 300 mg that is givenin combination with palbociclib may be reduced by 50%. Accordingly, thedose may be given as 150 mg bid or 300 mg once daily.

Similarly, the normal recommended dose of palbociclib may be reducedwhen used in combination with RAD1901. The dose of palbociclib may bereduced and combined with the normal recommended monotherapy dose ofRAD1901 or a reduced RAD1901 dose wherein the reduced dose is 25% to 75%less than the normal recommended dose as exemplified immediately above.For example, a recommended dose of palbociclib of 125 mg qd might begiven as a qd dose of 25% to 75% less than the 125 mg dose. For example,125 mg of a recommended palbociclib dose may be reduced to a dose ofbetween 31.25 mg to 93.75 mg. In some embodiments, a specificpre-specified reduction dose of from 125 mg to 100 mg daily or from 125mg to 75 mg daily may be used. Alternatively, it may be desirable toreduce the frequency of palbociclib from a recommended 21 day out of 28day on cycle to some amount less. For example, the dosing frequency canbe reduced to 15 to 20 days to out of a 28 day cycle or to 14 days outof a 28 day cycle, or the dosing frequency may be reduced to 8 days to13 days out of a 28 day cycle or to 7 days out of a 28 day cycle. Thedays dosed may be consecutive or combined as needed under thecircumstance. In one embodiment, the total dose over a dosing intervalis reduced by 25% to 75% of the recommended dose and that reduction maycome as a result of less frequent dosing, reduced dosage or acombination thereof. For example, a recommended dosing cycle of 28 daysof palbociclib (21 days at a dose of 125 mg qd) results in a total doseover 28 days of 2,625 mg (21 dosing days times 125 mg total per day).This amount can be reduced to between from 656.25 mg per 28 day cycle to1,968.75 mg per 28 day cycle. In another embodiment, a recommended 28day total cycle dose of 2,625 mg be reduced to 2,100 mg per 28 daycycle.

In certain embodiments, a therapeutically effective amount of thecombination may utilize a therapeutically effective amount of eithercompound administered alone. In other embodiments, due to thesignificantly improved, synergistic therapeutic effect achieved by thecombination, the therapeutically effective amounts of RAD1901 orsolvates (e.g., hydrate) or salts thereof and the CDK4 and/or CDK6inhibitor(s) as described herein (e.g., ribociclib, abemaciclib andpalbociclib) when administered in the combination may be smaller thanthe therapeutically effective amounts of RAD1901 or solvates (e.g.,hydrate) or salts thereof and the CDK4 and/or CDK6 inhibitor(s) asdescribed herein (e.g., ribociclib, abemaciclib and palbociclib)required when administered alone; and one or both compounds may beadministered at a dosage that is lower than the dosage at which theywould normally be administered when given separately. Without beingbound by any specific theory, the combination therapy achieves asignificantly improved effect by reducing the dosage of at least one orall of RAD1901 or solvates (e.g., hydrate) or salts thereof and the CDK4and/or CDK6 inhibitor(s) as described herein (e.g., ribociclib,abemaciclib and palbociclib), thereby eliminating or alleviatingundesirable toxic side effects.

In some embodiments, the therapeutically effective amount of RAD1901 orsolvates (e.g., hydrate) or salts thereof when administered as part ofthe combination is about 30% to about 200%, about 40% to about 200%,about 50% to about 200%, about 60% to about 200%, about 70% to about200%, about 80% to about 200%, about 90% to about 200%, about 100% toabout 200%, 30% to about 150%, about 40% to about 150%, about 50% toabout 150%, about 60% to about 150%, about 70% to about 150%, about 80%to about 150%, about 90% to about 150%, about 100% to about 150%, about30% to about 120%, about 40% to about 120%, about 50% to about 120%,about 60% to about 120%, about 70% to about 120%, about 80% to about120%, about 90% to about 120%, about 100% to about 120%, 30% to about110%, about 40% to about 110%, about 50% to about 110%, about 60% toabout 110%, about 70% to about 110%, about 80% to about 110%, about 90%to about 110%, or about 100% to about 110% of the therapeuticallyeffective amount of RAD1901 or solvates (e.g., hydrate) or salts thereofwhen administered alone. In some embodiments, the therapeuticallyeffective amount of the CDK4 and/or CDK6 inhibitor as described herein(e.g., ribociclib, abemaciclib and palbociclib) when administered aspart of the combination is about 30% to about 200%, about 40% to about200%, about 50% to about 200%, about 60% to about 200%, about 70% toabout 200%, about 80% to about 200%, about 90% to about 200%, about 100%to about 200%, 30% to about 150%, about 40% to about 150%, about 50% toabout 150%, about 60% to about 150%, about 70% to about 150%, about 80%to about 150%, about 90% to about 150%, about 100% to about 150%, about30% to about 120%, about 40% to about 120%, about 50% to about 120%,about 60% to about 120%, about 70% to about 120%, about 80% to about120%, about 90% to about 120%, about 100% to about 120%, 30% to about110%, about 40% to about 110%, about 50% to about 110%, about 60% toabout 110%, about 70% to about 110%, about 80% to about 110%, about 90%to about 110%, or about 100% to about 110% of the therapeuticallyeffective amount of the CDK4 and/or CDK6 inhibitor as described herein(e.g., ribociclib, abemaciclib and palbociclib) when administered alone.

In certain embodiments, the cancers or tumors are resistant ER-drivencancers or tumors (e.g. having mutant ER binding domains (e.g. ERαcomprising one or more mutations including, but not limited to, Y537X₁wherein X₁ is S, N, or C, D538G, L536X₂ wherein X₂ is R or Q, P535H,V534E, S463P, V392I, E380Q and combinations thereof), overexpressors ofthe ERs or tumor and/or cancer proliferation becomes ligand independent,or tumors and/or cancers that progress with treatment of another SERD(e.g., fulvestrant, TAS-108 (SR16234), ZK191703, RU58668, GDC-0810(ARN-810), GW5638/DPC974, SRN-927, ICI182782 and AZD9496), Her2inhibitors (e.g., trastuzumab, lapatinib, ado-trastuzumab emtansine,and/or pertuzumab), chemo therapy (e.g., abraxane, adriamycin,carboplatin, cytoxan, daunorubicin, doxil, ellence, fluorouracil,gemzar, helaven, lxempra, methotrexate, mitomycin, micoxantrone,navelbine, taxol, taxotere, thiotepa, vincristine, and xeloda),aromatase inhibitor (e.g., anastrozole, exemestane, and letrozole),selective estrogen receptor modulators (e.g., tamoxifen, raloxifene,lasofoxifene, and/or toremifene), angiogenesis inhibitor (e.g.,bevacizumab), and/or rituximab.

In certain embodiments, the dosage of RAD1901 or solvates (e.g.,hydrate) or salts thereof in a combination with a CDK4 and/or CDK6inhibitor as described herein (e.g., ribociclib, abemaciclib andpalbociclib) for use in the presently disclosed methods general for anadult subject may be approximately 30 mg to 2,000 mg, 100 mg to 1,500mg, or 150 mg to 1,500 mg p.o., q.d. This daily dosage may be achievedvia a single administration or multiple administrations.

A combination of one or more CDK4 and/or CDK6 inhibitor(s) as describedherein (e.g., ribociclib, abemaciclib and palbociclib) and RAD1901 orsolvates (e.g., hydrate) or salts thereof may be administered to asubject one time or multiple times. In those embodiments wherein thecompounds are administered multiple times, they may be administered at aset interval, e.g., daily, every other day, weekly, or monthly.Alternatively, they can be administered at an irregular interval, forexample on an as-needed basis based on symptoms, patient health, and thelike.

(3) Formulation

In some embodiments, RAD1901 or solvates (e.g., hydrate) or saltsthereof and the CDK4 and/or CDK6 inhibitor(s) as described herein (e.g.,ribociclib, abemaciclib and palbociclib) are administered in separateformulations. In certain of these embodiments, the formulations may beof the same type. For example, both formulations may be designed fororal administration (e.g., via two separate pills) or for injection(e.g., via two separate injectable formulations). In other embodiments,RAD1901 or solvates (e.g., hydrate) or salts thereof and the CDK4 and/orCDK6 inhibitor(s) as described herein (e.g., ribociclib, abemaciclib andpalbociclib) may be formulated in different types of formulations. Forexample, one compound may be in a formulation designed for oraladministration, while the other is in a formulation designed forinjection.

In other embodiments, RAD1901 or solvates (e.g., hydrate) or saltsthereof and the CDK4 and/or CDK6 inhibitor(s) (e.g., ribociclib,abemaciclib and palbociclib) described herein are administered as partof a single formulation. For example, RAD1901 or solvates (e.g.,hydrate) or salts thereof and the CDK4 and/or CDK6 inhibitor(s) asdescribed herein (e.g., ribociclib, abemaciclib and palbociclib) areformulated in a single pill for oral administration or in a single dosefor injection. Provided herein in certain embodiments are combinationformulations comprising RAD1901 or solvates (e.g., hydrate) or saltsthereof and one or more CDK4 and/or CDK6 inhibitor(s) as describedherein (e.g., ribociclib, abemaciclib and palbociclib). In certainembodiments, administration of the compounds in a single formulationimproves patient compliance.

The therapeutically effective amount of each compound when administeredin combination may be lower than the therapeutically effective amount ofeach compound administered alone.

In some embodiments, a formulation comprising RAD1901 or solvates (e.g.,hydrate) or salts thereof, one or more to the CDK4 and/or CDK6inhibitor(s) (e.g., ribociclib, abemaciclib and palbociclib), or bothRAD1901 or solvates (e.g., hydrate) or salts thereof and the one or moreCDK4 and/or CDK6 inhibitor(s) (e.g., ribociclib, abemaciclib andpalbociclib) may further comprise one or more pharmaceutical excipients,carriers, adjuvants, and/or preservatives.

The RAD1901 or solvates (e.g., hydrate) or salts thereof and the CDK4and/or CDK6 inhibitor(s) (e.g., ribociclib, abemaciclib and palbociclib)for use in the presently disclosed methods can be formulated into unitdosage forms, meaning physically discrete units suitable as unitarydosage for subjects undergoing treatment, with each unit containing apredetermined quantity of active material calculated to produce thedesired therapeutic effect, optionally in association with a suitablepharmaceutical carrier. The unit dosage form can be for a single dailydose or one of multiple daily doses (e.g., about 1 to 4 or more timesq.d.). When multiple daily doses are used, the unit dosage form can bethe same or different for each dose. In certain embodiments, thecompounds may be formulated for controlled release.

The RAD1901 or solvates (e.g., hydrate) or salts thereof and salts orsolvates and the CDK4 and/or CDK6 inhibitor(s) (e.g., ribociclib,abemaciclib and palbociclib) for use in the presently disclosed methodscan be formulated according to any available conventional method.Examples of preferred dosage forms include a tablet, a powder, a subtlegranule, a granule, a coated tablet, a capsule, a syrup, a troche, aninhalant, a suppository, an injectable, an ointment, an ophthalmicointment, an eye drop, a nasal drop, an ear drop, a cataplasm, a lotionand the like. In the formulation, generally used additives such as adiluent, a binder, an disintegrant, a lubricant, a colorant, a flavoringagent, and if necessary, a stabilizer, an emulsifier, an absorptionenhancer, a surfactant, a pH adjuster, an antiseptic, an antioxidant andthe like can be used. In addition, the formulation is also carried outby combining compositions that are generally used as a raw material forpharmaceutical formulation, according to the conventional methods.Examples of these compositions include, for example, (1) an oil such asa soybean oil, a beef tallow and synthetic glyceride; (2) hydrocarbonsuch as liquid paraffin, squalane and solid paraffin; (3) ester oil suchas octyldodecyl myristic acid and isopropyl myristic acid; (4) higheralcohol such as cetostearyl alcohol and behenyl alcohol; (5) a siliconresin; (6) a silicon oil; (7) a surfactant such as polyoxyethylene fattyacid ester, sorbitan fatty acid ester, glycerin fatty acid ester,polyoxyethylene sorbitan fatty acid ester, a solid polyoxyethylenecastor oil and polyoxyethylene polyoxypropylene block co-polymer; (8)water soluble macromolecule such as hydroxyethyl cellulose, polyacrylicacid, carboxyvinyl polymer, polyethyleneglycol, polyvinylpyrrolidone andmethylcellulose; (9) lower alcohol such as ethanol and isopropanol; (10)multivalent alcohol such as glycerin, propyleneglycol, dipropyleneglycoland sorbitol; (11) a sugar such as glucose and cane sugar; (12) aninorganic powder such as anhydrous silicic acid, aluminum magnesiumsilicate and aluminum silicate; (13) purified water, and the like.Additives for use in the above formulations may include, for example, 1)lactose, corn starch, sucrose, glucose, mannitol, sorbitol, crystallinecellulose and silicon dioxide as the diluent; 2) polyvinyl alcohol,polyvinyl ether, methyl cellulose, ethyl cellulose, gum arabic,tragacanth, gelatine, shellac, hydroxypropyl cellulose,hydroxypropylmethyl cellulose, polyvinylpyrrolidone, polypropyleneglycol-poly oxyethylene-block co-polymer, meglumine, calcium citrate,dextrin, pectin and the like as the binder; 3) starch, agar, gelatinepowder, crystalline cellulose, calcium carbonate, sodium bicarbonate,calcium citrate, dextrin, pectic, carboxymethylcellulose/calcium and thelike as the disintegrant; 4) magnesium stearate, talc,polyethyleneglycol, silica, condensed plant oil and the like as thelubricant; 5) any colorants whose addition is pharmaceuticallyacceptable is adequate as the colorant; 6) cocoa powder, menthol,aromatizer, peppermint oil, cinnamon powder as the flavoring agent; 7)antioxidants whose addition is pharmaceutically accepted such asascorbic acid or alpha-tophenol.

The one or more CDK4 and/or CDK6 inhibitor(s) as described herein (e.g.,ribociclib, abemaciclib and palbociclib) and RAD1901 or solvates (e.g.,hydrate) or salts thereof for use in the presently disclosed methods canbe formulated into a pharmaceutical composition as any one or more ofthe active compounds described herein and a physiologically acceptablecarrier (also referred to as a pharmaceutically acceptable carrier orsolution or diluent). Such carriers and solutions includepharmaceutically acceptable salts and solvates of compounds used in themethods of the instant invention, and mixtures comprising two or more ofsuch compounds, pharmaceutically acceptable salts of the compounds andpharmaceutically acceptable solvates of the compounds. Such compositionsare prepared in accordance with acceptable pharmaceutical proceduressuch as described in Remington's Pharmaceutical Sciences, 17th edition,ed. Alfonso R. Gennaro, Mack Publishing Company, Eaton, Pa. (1985),which is incorporated herein by reference.

The term “pharmaceutically acceptable carrier” refers to a carrier thatdoes not cause an allergic reaction or other untoward effect in patientsto whom it is administered and are compatible with the other ingredientsin the formulation. Pharmaceutically acceptable carriers include, forexample, pharmaceutical diluents, excipients or carriers suitablyselected with respect to the intended form of administration, andconsistent with conventional pharmaceutical practices. For example,solid carriers/diluents include, but are not limited to, a gum, a starch(e.g., corn starch, pregelatinized starch), a sugar (e.g., lactose,mannitol, sucrose, dextrose), a cellulosic material (e.g.,microcrystalline cellulose), an acrylate (e.g., polymethylacrylate),calcium carbonate, magnesium oxide, talc, or mixtures thereof.Pharmaceutically acceptable carriers may further comprise minor amountsof auxiliary substances such as wetting or emulsifying agents,preservatives or buffers, which enhance the shelf life or effectivenessof the therapeutic agent.

The one or more CDK4 and/or CDK6 inhibitor(s) as described herein (e.g.,ribociclib, abemaciclib and palbociclib) and RAD1901 or solvates (e.g.,hydrate) or salts thereof in a free form can be converted into a salt byconventional methods. The term “salt” used herein is not limited as longas the salt is formed with RAD1901 or solvates (e.g., hydrate) or saltsthereof and is pharmacologically acceptable; preferred examples of saltsinclude a hydrohalide salt (for instance, hydrochloride, hydrobromide,hydroiodide and the like), an inorganic acid salt (for instance,sulfate, nitrate, perchlorate, phosphate, carbonate, bicarbonate and thelike), an organic carboxylate salt (for instance, acetate salt, maleatesalt, tartrate salt, fumarate salt, citrate salt and the like), anorganic sulfonate salt (for instance, methanesulfonate salt,ethanesulfonate salt, benzenesulfonate salt, toluenesulfonate salt,camphorsulfonate salt and the like), an amino acid salt (for instance,aspartate salt, glutamate salt and the like), a quaternary ammoniumsalt, an alkaline metal salt (for instance, sodium salt, potassium saltand the like), an alkaline earth metal salt (magnesium salt, calciumsalt and the like) and the like. In addition, hydrochloride salt,sulfate salt, methanesulfonate salt, acetate salt and the like arepreferred as “pharmacologically acceptable salt” of the compoundsaccording to the present invention.

Isomers of RAD1901 or solvates (e.g., hydrate) or salts thereof and/orthe CDK4 and/or CDK6 inhibitor(s) (e.g., ribociclib, abemaciclib andpalbociclib) disclosed herein (e.g., geometric isomers, optical isomers,rotamers, tautomers, and the like) can be purified using generalseparation means, including for example recrystallization, opticalresolution such as diastereomeric salt method, enzyme fractionationmethod, various chromatographies (for instance, thin layerchromatography, column chromatography, glass chromatography and thelike) into a single isomer. The term “a single isomer” herein includesnot only an isomer having a purity of 100%, but also an isomercontaining an isomer other than the target, which exists even throughthe conventional purification operation. A crystal polymorph sometimesexists for RAD1901 or solvates (e.g., hydrate) or salts thereof and/or aCDK4 and/or CDK6 inhibitor (e.g., ribociclib, abemaciclib andpalbociclib), and all crystal polymorphs thereof are included in thepresent invention. The crystal polymorph is sometimes single andsometimes a mixture, and both are included herein.

In certain embodiments, RAD1901 or solvates (e.g., hydrate) or saltsthereof and/or CDK4 and/or CDK6 inhibitor (e.g., ribociclib, abemacicliband palbociclib) may be in a prodrug form, meaning that it must undergosome alteration (e.g., oxidation or hydrolysis) to achieve its activeform. Alternative, RAD1901 or solvates (e.g., hydrate) or salts thereofand/or CDK4 and/or CDK6 inhibitor (e.g., ribociclib, abemaciclib andpalbociclib) may be a compound generated by alteration of a parentalprodrug to its active form.

(4) Administration Route

Administration routes of RAD1901 or solvates (e.g., hydrate) or saltsthereof and/or CDK4 and/or CDK6 inhibitor(s) (e.g., ribociclib,abemaciclib and palbociclib) disclosed herein include but not limited totopical administration, oral administration, intradermal administration,intramuscular administration, intraperitoneal administration,intravenous administration, intravesical infusion, subcutaneousadministration, transdermal administration, and transmucosaladministration.

(5) Gene Profiling

In certain embodiments, the methods of tumor growth inhibition or tumorregression provided herein further comprise gene profiling the subject,wherein the gene to be profiled is one or more genes selected from thegroup consisting of ABL1, AKT1, AKT2, ALK, APC, AR, ARID1A, ASXL1, ATM,AURKA, BAP, BAP1, BCL2L11, BCR, BRAF, BRCA1, BRCA2, CCND1, CCND2, CCND3,CCNE1, CDH1, CDK4, CDK6, CDK8, CDKN1A, CDKN1B, CDKN2A, CDKN2B, CEBPA,CTNNB1, DDR2, DNMT3A, E2F3, EGFR, EML4, EPHB2, ERBB2, ERBB3, ESR1,EWSR1, FBXW7, FGF4, FGFR1, FGFR2, FGFR3, FLT3, FRS2, HIF1A, HRAS, IDH1,IDH2, IGF1R, JAK2, KDM6A, KDR, KIF5B, KIT, KRAS, LRP1B, MAP2K1, MAP2K4,MCL1, MDM2, MDM4, MET, MGMT, MLL, MPL, MSH6, MTOR, MYC, NF1, NF2,NKX2-1, NOTCH1, NPM, NRAS, PDGFRA, PIK3CA, PIK3R1, PML, PTEN, PTPRD,RARA, RB1, RET, RICTOR, ROS1, RPTOR, RUNX1, SMAD4, SMARCA4, SOX2, STK11,TET2, TP53, TSC1, TSC2, and VHL.

In some embodiments, this invention provides a method of treating asubpopulation of breast cancer patients wherein said sub-population hasincreased expression of one or more of the genes disclosed supra, andtreating said sub-population with an effective dose of a combination ofone or more CDK4 and/or CDK6 inhibitor(s) as described herein (e.g.,ribociclib, abemaciclib and palbociclib) and RAD1901 or solvates (e.g.,hydrate) or salts thereof according to the dosing embodiments asdescribed in this disclosure.

(6) Dose Adjusting

In addition to establishing the ability of RAD1901 to inhibit tumorgrowth, the results provided herein show that RAD1901 inhibits estradiolbinding to ER in the uterus and pituitary (Example III(A)). In theseexperiments, estradiol binding to ER in uterine and pituitary tissue wasevaluated by FES-PET imaging. After treatment with RAD1901, the observedlevel of ER binding was at or below background levels. These resultsestablish that the antagonistic effect of RAD1901 on ER activity can beevaluated using real-time scanning. Based on these results, methods areprovided herein for monitoring the efficacy of treatment RAD1901 orsolvates (e.g., hydrate) or salts thereof in a combination therapydisclosed herein by measuring estradiol-ER binding in one or more targettissues, wherein a decrease or disappearance in binding indicatesefficacy.

Further provided are methods of adjusting the dosage of RAD1901 orsolvates (e.g., hydrate) or salts thereof in a combination therapydisclosed herein based on estradiol-ER binding. In certain embodimentsof these methods, binding is measured at some point following one ormore administrations of a first dosage of the compound. If estradiol-ERbinding is not affected or exhibits a decrease below a predeterminedthreshold (e.g., a decrease in binding versus baseline of less than 5%,less than 10%, less than 20%, less than 30%, or less than 50%), thefirst dosage is deemed to be too low. In certain embodiments, thesemethods comprise an additional step of administering an increased seconddosage of the compound. These steps can be repeated, with dosagerepeatedly increased until the desired reduction in estradiol-ER bindingis achieved. In certain embodiments, these steps can be incorporatedinto the methods of inhibiting tumor growth provided herein. In thesemethods, estradiol-ER binding can serve as a proxy for tumor growthinhibition, or a supplemental means of evaluating growth inhibition. Inother embodiments, these methods can be used in conjunction with theadministration of RAD1901 or solvates (e.g., hydrate) or salts thereoffor purposes other than inhibition of tumor growth, including forexample inhibition of cancer cell proliferation.

In certain embodiments, the methods provided herein for adjusting thedosage of RAD1901 or salt or solvate (e.g., hydrate) thereof in acombination therapy comprise:

-   -   (1) administering a first dosage of RAD1901 or salt or solvate        (e.g., hydrate) thereof (e.g., about 350 to about 500 or about        200 to about 600 mg/day) for 3, 4, 5, 6, or 7 days;    -   (2) detecting estradiol-ER binding activity, for example using        FES-PET imaging as disclosed herein; wherein:        -   (i) if the ER binding activity is not detectable or is below            a predetermined threshold level, continuing to administer            the first dosage (i.e., maintain the dosage level); or        -   (ii) if the ER binding activity is detectable or is above a            predetermined threshold level, administering a second dosage            that is greater than the first dosage (e.g., the first            dosage plus about 50 to about 200 mg) for 3, 4, 5, 6, or 7            days, then proceeding to step (3);    -   (3) detecting estradiol-ER binding activity, for example using        FES-PET imaging as disclosed herein; wherein        -   (i) if the ER binding activity is not detectable or is below            a predetermined threshold level, continuing to administer            the second dosage (i.e., maintain the dosage level); or        -   (ii) if the ER binding activity is detectable or is above a            predetermined threshold level, administering a third dosage            that is greater than the second dosage (e.g., the second            dosage plus about 50 to about 200 mg) for 3, 4, 5, 6, or 7            days, then proceeding to step (4);    -   (4) repeating the steps above through a fourth dosage, fifth        dosage, etc., until no ER binding activity is detected.

In certain embodiments, the invention includes the use of PET imaging todetect and/or dose ER sensitive or ER resistant cancers.

(7) Combinations for the Methods Disclosed Herein

Another aspect of the invention relates to a pharmaceutical compositioncomprising RAD1901 or solvates (e.g., hydrate) or salts thereof and/orCDK4 and/or CDK6 inhibitor(s) (e.g., ribociclib, abemaciclib andpalbociclib) disclosed herein in a therapeutically effective amount asdisclosed herein for the combination methods set forth herein.

RAD1901-ERα Interactions

(1) Mutant ERα in ER Positive Breast Tumor Samples from Patients WhoReceived at Least One Line of Endocrine Treatment

In five studies reported in the past two years, a total of 187metastatic ER positive breast tumor samples from patients who receivedat least one line of endocrine treatment were sequenced and ER LBDmutations were identified in 39 patients (21%) (Jeselsohn). Among the 39patients, the six most frequent LBD mutations are shown in FIG. 39adapted from Jeselsohn.

The frequency of all LBD mutations are summarized in Table 9.

Computer modeling showed that RAD1901-ERα interactions are not likely tobe affected by mutants of LBD of ERα, e.g., Y537X mutant wherein X wasS, N, or C; D538G; and S463P, which account for about 81.7% of LBDmutations found in a recent study of metastatic ER positive breast tumorsamples from patients who received at least one line of endocrinetreatment (Table 10, Example V).

Provided herein are complexes and crystals of RAD1901 bound to ERαand/or a mutant ERα, the mutant ERα comprises one or more mutationsincluding, but not limited to, Y537X₁ wherein X₁ is S, N, or C, D538G,L536X₂ wherein X₂ is R or Q, P535H, V534E, S463P, V392I, E380Q andcombinations thereof.

In certain embodiments of the methods provided herein, the LBD of ERαand a mutant ERα comprises AF-2. In other embodiments, the LBDcomprises, consists of, or consists essentially of amino acids 299-554of ERα. In certain embodiments, the LBD of the mutant ERα comprises oneor more mutations including, but not limited to, Y537X₁ wherein X₁ is S,N, or C, D538G, L536X₂ wherein X₂ is R or Q, P535H, V534E, S463P, V392I,E380Q and combinations thereof. The term “and/or” as used hereinincludes both the “and” case and the “or” case.

The following examples are provided to better illustrate the claimedinvention and are not to be interpreted as limiting the scope of theinvention. To the extent that specific materials are mentioned, it ismerely for purposes of illustration and is not intended to limit theinvention. One skilled in the art may develop equivalent means orreactants without the exercise of inventive capacity and withoutdeparting from the scope of the invention. It will be understood thatmany variations can be made in the procedures herein described whilestill remaining within the bounds of the present invention. It is theintention of the inventors that such variations are included within thescope of the invention.

EXAMPLES

Materials and Methods

Test Compounds

RAD1901 used in the examples below was(6R)-6-(2-(N-(4-(2-(ethylamino)ethyl)benzyl)-N-ethylamino)-4-methoxyphenyl)-5,6,7,8-tetrahydronaphthalen-2-oldihydrochloride, manufactured by IRIX Pharmaceuticals, Inc. (Florence,S.C.). RAD1901 was stored as a dry powder, formulated for use as ahomogenous suspension in 0.5% (w/v) methylcellulose in deionized water,and for animal models was administered p.o. Tamoxifen, raloxifene andestradiol (E2) were obtained from Sigma-Aldrich (St. Louis, Mo.), andadministered by subcutaneous injection. Fulvestrant was obtained fromTocris Biosciences (Minneapolis, Minn.) and administered by subcutaneousinjection. Other laboratory reagents were purchased from Sigma-Aldrichunless otherwise noted.

Cell Lines

MCF-7 cells (human mammary metastatic adenocarcinoma) were purchasedfrom American Type Culture Collection (Rockville, Md.) and wereroutinely maintained in phenol red-free minimal essential medium (MEM)containing 2 mM L-glutamine and Earle's BSS, 0.1 mM non-essential aminoacids and 1 mM sodium pyruvate supplemented with 0.01 mg/ml bovineinsulin and 10% fetal bovine serum (Invitrogen, Carlsbad, Calif.), at 5%CO₂.

T47D cells were cultured in 5% CO₂ incubator in 10 cm dishes toapproximately 75% confluence in RPMI growth media supplemented with 10%FBS and 5 μg/mL human insulin.

In Vivo Xenograft Models

All mice were housed in pathogen-free housing in individually ventilatedcages with sterilized and dust-free bedding cobs, access to sterilizedfood and water ad libitum, under a light dark cycle (12-14 hourcircadian cycle of artificial light) and controlled room temperature andhumidity. Tumors were measured twice weekly with Vernier calipers andvolumes were calculated using the formula: (L*W²)*0.52.

PDx Models

Some examples of patient-derived xenograft models (PDx models) are shownin FIG. 1. PDx models with patient derived breast cancer tumor wereestablished from viable human tumor tissue or fluid that had beenserially passaged in animals (athymic nude mice (Nu (NCF)-Foxn1nu)) alimited number of times to maintain tumor heterogeneity. Pre-study tumorvolumes were recorded for each experiment beginning approximately oneweek prior to its estimated start date. When tumors reached theappropriate Tumor Volume Initiation (TVI) range (150-250 mm³), animalswere randomized into treatment and control groups and dosing initiated(Day 0, 8-10 subjects in each group); animals in all studies followedindividually throughout each experiment. Initial dosing began Day 0;animals in all groups were dosed by weight (0.01 mL per gram; 10 ml/kg).Each group was treated with vehicle (control, p.o., q.d. to theendpoint), tamoxifen (1 mg/subject, s.c., q.o.d. to the end point),fulvestrant (Faslodex®; 1 mg/subject or 3 mg/subject as needed, s.c.,qwk, ×5 and extended if necessary), or RAD1901 (30, 60 or 120 mg/kg ofthe subject, p.o., q.d. to the endpoint) as specified from day 0. Thetreatment period lasted for 56-60 days depending on the models. Thedrinking water for these PDx models was supplemented with 17β-estradiol.

Agent Efficacy

For all studies, beginning Day 0, tumor dimensions were measured bydigital caliper and data including individual and mean estimated tumorvolumes (Mean TV±SEM) recorded for each group; tumor volume wascalculated using the formula (Yasui et al. Invasion Metastasis17:259-269 (1997), which is incorporated herein by reference):TV=width²×length×0.52. Each group or study was ended once the estimatedgroup mean tumor volume reached the Tumor Volume (TV) endpoint (timeendpoint was 60 days; and volume endpoint was group mean 2 cm³);individual mice reaching a tumor volume of 2 cm³ or more were removedfrom the study and the final measurement included in the group meanuntil the mean reached volume endpoint or the study reached timeendpoint.

Efficacy Calculations and Statistical Analysis

% Tumor Growth Inhibition (% TGI) values were calculated at a singletime point (when the control group reached tumor volume or timeendpoint) and reported for each treatment group (T) versus control (C)using initial (i) and final (f) tumor measurements by the formula(Corbett T H et al. In vivo methods for screening and preclinicaltesting. In: Teicher B, ed., Anticancer Drug Development Guide. Totowa,N.J.: Humana. 2004: 99-123): % TGI=1−Tf−Ti/Cf−Ci.

Statistics

TGI Studies—One way ANOVA+Dunnett's Multiple Comparisons Test (Corbett TH et al).

Sample Collection

At endpoint, tumors were removed. One fragment was flash frozen, whileanother fragment was placed in 10% NBF for at least 24 hours andformalin fixed paraffin embedded (FFPE). Flash frozen samples werestored at −80° C.; FFPE blocks were stored at room temperature.

Western Blot

Cells were harvested and protein expression was analyzed using standardpractice. Tumors were harvested at the indicated time points after thelast day of dosing, homogenized in RIPA buffer with protease andphosphatase inhibitors using a Tissuelyser (Qiagen). Equal amounts ofprotein were separated by MW, transferred to nitrocellulose membranesand blotted with the following antibodies using standard practice:

-   -   Estrogen receptor (SantaCruz (HC-20); sc-543)    -   Progesterone receptor (Cell Signaling Technologies; 3153)    -   Vinculin (Sigma-Aldrich, v9131)

qPCR analyses were performed as follows: Cells were harvested, mRNA wasextracted, and equal amounts used for cDNA synthesis and qPCR withprimers specific for progesterone receptor, GREB1, and TFF1 (LifeTech).Bands were quantified using 1D Quant software (GE).

Immunohistochemistry

Tumors were harvested, formalin-fixed and embedded into paraffin.Embedded tumors were sectioned (6 μM) and stained with antibodiesspecific for ER, PR, and Her2. Quantitation was performed as follows:Five fields were counted for positive cells (0-100%) and intensity ofstaining (0-3+). H-scores (0-300) were calculated using the followingformula: % positivity*intensity.

Example I. RAD1901-Palbo Combinations Provided Enhanced Tumor GrowthInhibition in Tumor and/or Cancer Expressing WT ER or Mutant ER (e.g.,Y5378), with Different Prior Endocrine Therapy

I(A). Effectiveness of RAD1901 on Animal Xenografts Models

I(A)(i) RAD1901-Palbo Combinations Demonstrated Improved Tumor GrowthInhibition in PDx Models (PDx-1 to PDx-12) Regardless of ER Status andPrior Endocrine Therapy

FIG. 1 demonstrates tumor growth inhibition effects in various PDxmodels for mice treated with RAD1901 alone and/or a RAD1901-palbocombination. Twelve patient-derived xenograft models were screened totest RAD1901 response in a variety of genetic backgrounds with variedlevels of ER, PR and Her2. Full efficacy study was carried out for PDxmodels marked with “*” (PDx-1 to PDx-4, and PDx-12), with n=8-10. ThesePDx models were treated with vehicle (negative control), RAD1901 at adosage of 60 mg/kg p.o., q.d., or a RAD1901-palbo combination with 60mg/kg RAD1901 and palbociclib p.o., q.d. for 60 days. Screen study wascarried out for other PDx models (PDx-5 to PDx-11), with n=3 at treatedwith vehicle (negative control) or RAD1901 at a dosage of 90 mg/kg, for60 days, p.o., q.d. As demonstrated in FIG. 1, PDx models in which thegrowth was driven by ER and an additional driver (e.g., PR+ and/orHer2+) benefited from the RAD1901 treatments. RAD1901 was efficacious ininhibiting tumor growth in models with ER mutations and/or high levelexpression of Her2 (PDx), regardless of prior treatment, eithertreatment naïve (Rx-naïve), or treated with aromatase inhibitor,tamoxifen (tam), chemotherapy (chemo), Her2 inhibitors (Her2i, e.g.,trastuzumab, lapatinib), bevacizumab, fulvestrant, and/or rituximab.

RAD1901-palbo combinations demonstrated enhanced tumor growth inhibitionin PDx models in which RAD1901 single agent treatment achieved TGI of64% or lower (PDx-2, PDx-5, PDx-7, PDx-8, PDx-9, and PDx-10). Said PDxmodels include treatment naïve models (PDx-2, ER++, PR++ and Her2+), andmodels with prior treatments of aromatase inhibitor (AI, tamoxifen(tam), chemotherapy (chemo), Her2 inhibitors (Her2i, e.g., trastuzumab,lapatinib), bevacizumab, fulvestrant, and/or rituximab (PDx-5, PDx-7,PDx-8, PDx-9, and PDx-10). PDx-5 models expressed mutant ESR1, whileother PDx models expressed WT ESR1. PDx-2, and PDx-5 models were PR+ andHer2+, while other PDx models were PR− and HER2+. Because RAD1901 singleagent treatment provided TGI of 65% or higher in PDx-6 and PDx-11models, the differences between RAD1901 treatment with and withoutpalbociclib could not be demonstrated in FIG. 1. See, e.g., Example(I)(A)(ii), and FIG. 3B showing RAD1901-palbo combinations caused moresignificant tumor regression than RAD1901 alone in PDx11 models.

I(A)(ii) RAD1901-Palbo Combination Drove More Regression than RAD1901Alone in Xenograft Models Expressing WT ER

I(A)(ii)(1) RAD1901-Palbo Drove More Regression than RAD1901 Alone inMCF-7 Xenografts that were Responsive to Fulvestrant Treatments.

MCF7 Xenograft Model

Two days before cell implantation, Balb/C-Nude mice were inoculated with0.18/90-day release 17β-estradiol pellets. MCF7 cells (PR+, Her2-) wereharvested and 1×10⁷ cells were implanted subcutaneously in the rightflank of Balb/C-Nude mice. When the tumors reached an average of 200mm³, the mice were randomized into treatment groups by tumor volume andtreated with the test compounds. Each group was treated with vehicle(control, p.o., q.d. to the endpoint), fulvestrant (Faslodex®; 3mg/subject, s.c., qwk×5 and extended if necessary), RAD1901 (30 mg/kg or60 mg/kg of the subject, p.o., q.d. to the endpoint), palbociclib (45mg/kg or 75 mg/kg or 100 mg/kg, p.o., q.d. to the end point), orRAD1901-palbo combination at doses specified from day 0. The treatmentperiod lasted for 28 days.

FIGS. 2A-C demonstrate that in MCF7 Xenograft Model, RAD1901-palbocombination with RAD1901 at 60 mg/kg p.o., q.d. and palbociclib at 45mg/kg p.o., q.d. RAD1901 (60 mg/kg p.o., q.d.) significantly reducedtumor size by about 50% by day 14. RAD1901 and palbociclib whenadministered alone exhibited efficacy in inhibiting tumor growth.

To confirm these results, MCF7 xenograft mice were treated with vehicle(negative control), RAD1901 (30 or 60 mg/kg, p.o., q.d), palbociclib (45mg/kg, p.o., q.d), a combination of RAD1901 (30 or 60 mg/kg, p.o., q.d)and palbociclib (45 mg/kg, p.o., q.d), fulvestrant (3 mg/dose, s.c.,qwk) or a combination of fulvestrant (3 mg/dose, s.c., qwk) andpalbociclib (45 mg/kg, p.o., q.d.). Tumor size was measured at varioustime points for 27 days.

Results are shown in FIGS. 2A-B. Treatment with the combination ofRAD1901 (60 mg/kg) and palbociclib (45 mg/kg) once again resulted insignificant tumor regression, with superior results to treatment withRAD1901, palbociclib, or fulvestrant alone, or with a combination offulvestrant and palbociclib (FIGS. 2A-B).

FIG. 2C demonstrates that RAD1901-palbo combinations with RAD1901 at adose of 30 mg/kg or 60 mg/kg both provided similar effects, althoughRAD1901 alone at 30 mg/kg was not as effective as RAD1901 alone at 60mg/kg in inhibiting tumor growth. Said results suggest a RAD1901-palbocombination with a lower dose of RAD1901 (e.g., 30 mg/kg) was sufficientto maximize the tumor growth inhibition/tumor regression effects in saidxenograft models.

Treatment with the combination of RAD1901 and palbociclib was also moreeffective at decreasing ER and PR expression in vivo in the MCF7xenograft models than treatment with RAD1901, palbociclib, orfulvestrant alone, or treatment with a combination of fulvestrant andpalbociclib (FIG. 13); tumors harvested two hours after the lastdosing).

I(A)(ii)(2) RAD1901-Palbo Drove More Tumor Regression than RAD1901 Alonein PDx-11 and PDx-2 Models that were Responsive to FulvestrantTreatments.

ER WT PDx models PDx-2 (PR+, Her2+, treatment naïve) and PDx-11 (PR+,Her2+, treated with AI, fulvestrant and chemo) exhibited differentsensitivities to fulvestrant (3 mg/dose, s.c., qwk). PDx-2 and PDx-11models were treated with a combination of RAD1901 (60 mg/kg, p.o., q.d.)and palbociclib (75 mg/kg, p.o., q.d.), RAD1901 alone (60 mg/kg, p.o.,q.d.), palbociclib alone (75 mg/kg, p.o., q.d.), or fulvestrant alone (3mg/dose, s.c., qwk). PDx-11 models were also treated with a combinationof palbociclib (75 mg/kg, p.o., q.d.), and fulvestrant (3 mg/dose, s.c.,qwk).

In PDx-11 models, administration of fulvestrant or palbociclib alonesignificantly inhibited tumor growth, with fulvestrant treated miceexhibiting better effects in tumor growth inhibition. The ful-palbocombination exhibited slight tumor regression. Unexpectedly,administration of RAD1901 alone or in combination with palbociclibresulted in a significant tumor regression, with the combinationachieved even more significant tumor regression effects in the wild-typeESR1 PDx models (FIG. 3B)).

In PDx-2 models, oral administration of RAD1901 alone achieved bettereffects of inhibiting tumor growth comparing to injection of fulvestrantalone (FIG. 4A). Furthermore, administration of RAD1901 or palbociclibalone significantly inhibited tumor growth. Unexpectedly, administrationof RAD1901 in combination with palbociclib resulted in even moreenhanced effect in inhibiting tumor growth (FIG. 4B)).

Furthermore, in PDx-4 model that were responsive to fulvestranttreatment (1 mg/dose, qwk, s.c.), RAD1901-mediated tumor growthinhibition was maintained in the absence of treatment at least twomonths after RAD1901 treatment (30 mg/kg, p.o., q.d.) period ended,while estradiol treatment continued (FIG. 5).

Thus, a RAD1901-palbo combination is likely to benefit a patient ininhibiting tumor growth after treatment ends, especially when CDK4/6inhibitors (e.g., ribociclib, abemaciclib and palbociclib) can only beadministered intermittently due to their side effects (O'Leary).

I(A)(iii) RAD1901-Palbo Drove More Regression than RAD1901 Alone inXenograft Models Expressing Mutant ER (ERα Y537S)

I(A)(iii)(1) RAD1901-Palbo Drove More Regression than RAD1901 Alone inPDx-5 Models that were Hardly Responsive to Fulvestrant Treatments.

PDx-5 models were prepared following similar protocol as described suprafor PDx models. The tumor sizes of each dosing group were measured twiceweekly with Vernier calipers, and volumes were calculated using theformula (L*W2)*0.52.

Inhibition of tumor growth by RAD1901 (60 mg/kg, p.o., q.d.),palbociclib (100 mg/kg, dropped to 75 mg/kg mid-study due totolerability issues, p.o., q.d.), and RAD1901 (60 mg/kg, p.o., q.d.) incombination with palbociclib (100 mg/kg, dropped to 75 mg/kg mid-studydue to tolerability issues) in mutant ER PDx-5 models (PDx models withpatient-derived breast cancer tumor having the Y537S estrogen receptormutation, PR+, Her2+, prior treatment with aromatase inhibitor) wasassessed using the method described supra. For tumors expressing certainERα mutations (e.g., Y537S), combination treatment of RAD1901 andpalbociclib was more effective in inhibiting tumor growth than treatmentwith either agent alone (FIG. 6A)). These PDx models were not sensitiveto fulvestrant (3 mg/dose) treatment (FIG. 6A). Combination treatment ofRAD1901 and palbociclib was more effective than treatment with eitheragent alone in driving tumor regressions in the RDx-5 models (FIGS.6B-C), showing the change of the individual tumor size from baseline onday 17 and day 56, respectively).

PDx-5 was treated with vehicle (negative control), fulvestrant (faslodex3 mg/dose, s.c., qwk), RAD1901 (60 mg/kg or 120 mg/kg, p.o., q.d.),palbociclib (100 mg/kg, p.o., q.d., dropped to 75 mg/kg mid-study due totolerability issues), or the combination of RAD1901 (60 or 120 mg/kg,p.o., q.d.) and palbociclib (100 mg/kg, p.o., q.d., dropped to 75 mg/kgmid-study due to tolerability issues). The tumor sizes measured wereshown in (FIGS. 7A-B). PDx-5 models were resistant to fulvestranttreatment, but RAD1901 and palbociclib either alone or in combinationinhibited tumor growth. At a dose of 60 mg/kg, RAD1901 alone hadsubstantially the same efficacy as palbociclib alone at 100 mg/kg (FIG.7A); while at a dose of 120 mg/kg, RAD1901 alone had an improvedefficacy comparing to palbociclib alone at 100 mg/kg (FIG. 7B). RAD1901at 60 mg/kg p.o. achieved significant inhibition of tumor growth (FIG.8). Furthermore, administration of palbociclib alone or in combinationwith fulvestrant significantly inhibited tumor growth in the mutant PDxmodel, although the combination did not further enhance the inhibition(FIG. 8).

Unexpectedly, RAD1901 alone or in combination with palbociclib achievedeven more significant tumor inhibition, with the combination almostcompletely inhibited tumor growth in the mutant PDx model; and aRAD1901-palbo combination with a lower dose of RAD1901 (e.g., 60 mg/kg)was sufficient to maximize the tumor growth inhibition/tumor regressioneffects in PDx-5 models (FIGS. 7A-B).

Thus, the results showed that RAD1901 was an effective endocrinebackbone that potentiated the tumor growth inhibition of targetedagents. Furthermore, RAD1901 showed potent anti-tumor activity in PDxmodels derived from patients that have had multiple prior endocrinetherapies including those that are insensitive to fulvestrant.

I(A)(iv) Pharmacokinetic Evaluation of Fulvestrant Treatments toNon-Tumor Bearing Mice.

Various doses of fulvestrant were administered to mice and demonstratedsignificant dose exposure to the subjects (Figure. 9).

Fulvestrant was administered at 1, 3, or 5 mg/dose subcutaneously tonude mice on day 1 (D1 Rx) and day 8 (D8 Rx, n=4/dose level). Blood wascollected at the indicated time points for up to 168 hours after thesecond dose, centrifuged, and plasma was analyzed by LiquidChromatography-Mass Spectrometry.

I(B) RAD1901 Promoted Survival in a Mouse Xenograft Model of BrainMetastasis (MCF-7 Intracranial Models).

The potential ability of RAD1901 to cross the blood-brain barrier andinhibit tumor growth was further evaluated using an MCF-7 intracranialtumor xenograft model.

Female athymic nude mice (Crl:NU(NCr)-Foxn1nu) were used for tumorxenograft studies. Three days prior to tumor cell implantation, estrogenpellets (0.36 mg E2, 60-day release, Innovative Research of America,Sarasota, Fla.) were implanted subcutaneously between the scapulae ofall test animals using a sterilized trochar. MCF-7 human breastadenocarcinoma cells were cultured to mid-log phase in RPMI-1640 mediumcontaining 10% fetal bovine serum, 100 units/mL penicillin G, 100 μg/mLstreptomycin sulfate, 2 mM glutamine, 10 mM HEPES, 0.075% sodiumbicarbonate and 25 g/mL gentamicin. On the day of tumor cell implant,the cells were trypsinized, pelleted, and resuspended in phosphatebuffered saline at a concentration of 5×10⁷ cells/mL. Each test mousereceived 1×10⁶ MCF-7 cells implanted intracranially.

Five days after tumor cell implantation (designated as day 1 of thestudy), mice were randomized into three groups of 12 animals each andtreated with vehicle, fulvestrant (0.5 mg/animal q.d.), or RAD1901 (120mg/kg q.d.), as described above.

The endpoint was defined as a mortality or 3× survival of the controlgroup, whichever comes first. Treatment tolerability was assessed bybody weight measurements and frequent observation for clinical signs oftreatment-related adverse effects. Animals with weight loss exceeding30% for one measurement, or exceeding 25% for three measurements, werehumanely euthanized and classified as a treatment-related death.Acceptable toxicity was defined as a group-mean body weight loss of lessthan 20% during the study and not more than one treatment-related deathamong ten treated animals, or 10%. At the end of study animals wereeuthanized by terminal cardiac puncture under isoflurane anesthesia.RAD1901 and fulvestrant concentration in plasma and tumor weredetermined using LC-MS/MS.

Kaplan Meier survival analysis demonstrated that RAD1901 significantlyprolonged survival compared to fulvestrant (P<0.0001; FIG. 10). Noanimals in the control or fulvestrant group survived beyond day 20 andday 34 respectively, whereas 41% (5/12) of the RAD1901 treated animalssurvived until the end of the study on day 54.

Concentration of RAD1901 in the plasma was 738±471 ng/mL and in theintracranial tumor was 462±105 ng/g supporting the hypothesis thatRAD1901 is able to effectively cross the blood-brain barrier. Incontrast, concentrations of fulvestrant were substantially lower in theplasma (21±10 ng/mL) and in the intracranial tumor (8.3±0.8 ng/g).

Example II. RAD1901 Preferably Accumulated in Tumor and could beDelivered to Brain

MCF-7 xenografts as described in Example I(A)(i) were further evaluatedfor RAD1901 concentration in plasma and tumor using LC-MS/MS. At the endof study, the concentration of RAD1901 in plasma was 344±117 ng/mL andin tumor in 11,118±3,801 ng/mL for the 60 mg/kg dose level. A similartumor to plasma ratio was also observed at lower dose levels where tumorconcentrations were approximately 20-30 fold higher than in plasma.RAD1901 levels in plasma, tumor, and brain for mice treated for 40 daysare summarized in Table 1. A significant amount of RAD1901 was deliveredto the brain of the treated mice (e.g., see the B/P ratio (RAD1901concentration in brain/the RAD1901 concentration in plasma)), indicatingthat RAD1901 was able to cross the blood-brain barrier (BBB).Unexpectedly, RAD1901 preferably accumulated in the tumor. See, e.g.,the T/P (RAD1901 concentration in tumor/RAD1901 concentration in plasma)ratio shown in Table 1.

Example III. RAD1901 Inhibited ER Pathway and Degraded ER

III(A). RAD1901 Decreased ER-Engagements in Uterus and Pituitary inHealthy Postmenopausal Female Human Subjects.

The subjects had an amenorrhea duration of at least 12 months and serumFSH consistent with menopause. The subjects were 40-75 years old withBMI of 18.0-30 kg/m². Subjects had intact uterus. Subjects havingevidence of clinically relevant pathology, increased risk of stroke orof history venous thromboembolic events, or use of concomitantmedication less than 14 days prior to admission to clinical researchcenter (paracetamol allowed up to 3 days prior) were excluded.

FES-PET was performed at baseline and after 6 days of exposure toRAD1901 to evaluate ER engagement in the uterus. RAD1901 occupied 83%and 92% of ER in the uterus at the 200 mg (7 subjects) and 500 mg (6subjects) dose levels, respectively.

FES-PET imaging showed significant reduction in binding oflabeled-estradiol to both the uterus and pituitary after RAD1901treatment with 200 mg or 500 mg (p.o., q.d, 6 days).

Due to the high ER expression, the uterus showed a strong FES-PET signalat baseline before RAD1901 treatment (FIG. 11A), baseline transversalview for uterus FES-PET scan of Subject 3 treated with 200 mg doselevel; FIG. 11B, baseline sagittal view and transversal view for uterusFES-PET scan respectively of Subject 7 treated with 500 mg dose level).However, when scanned four hours post dosing on day 6 in the study, theuterus was hardly visible (at or close to background FES-PET signal(FIG. 11A), Day 6 transversal view for uterus scan of Subject 3; andFIG. 11B, Day 6 sagittal view and transversal view for uterus scanrespectively of Subject 7). Such data were consistent with ERdegradation and/or competition for the binding to the receptor. FIGS.11A-B also include CT scan of the uterus scanned by FES-PET showing theexistence of the uterus before and after RAD1901 treatment.

The FES-PET uterus scan results were further quantified to show thechange of post-dose ER-binding from baseline for 7 subjects (FIG.11(C)), showing Subjects 1-3 and Subjects 4-7 as examples of the 200 mgdose group and 500 mg dose group, respectively. RAD1901 showed robust ERengagement at the lower dose level (200 mg).

FIG. 12 showed a representative image of FES-PET scan of the uterus (A)and pituitary (B) before (Baseline) and after (Post-treatment) RAD1901treatment at 500 mg p.o., q.d., for six days. FIG. 12A showed theFES-PET scan of the uterus by (a) Lateral cross-section; (b) longitudecross-section; and (c) longitude cross-section.

The subject's post dose FES-PET scan of uterus and pituitary showed nonoticeable signal of ER binding at uterus (FIG. 12A, Post-treatment) andat pituitary (FIG. 12B, Post-treatment), respectively.

Thus, the results showed that RAD1901 effectively engaged ER in human ata dosage of 200 and 500 mg p.o., q.d., for six days.

Standard uptake value (SUV) for uterus, muscle and bone were calculatedand summarized for RAD1901 treatments at 200 mg and 500 mg p.o., q.d. inTables 2 and 3, respectively. Post-dose uterine signals were at or closeto levels from “non-target tissues,” suggesting a complete attenuationof FES-PET uptake post RAD1901 treatment. Almost no change was observedin pre-versus post-treatment PET scans in tissues that did notsignificant express estrogen receptor.

Thus, RAD1901 or salt or solvate (e.g., hydrate) thereof may be used intreating cancer and/or tumor cells having overexpression of ER (e.g.,breast cancer, uterus cancer, and ovary cancer), without negativeeffects to other organs (e.g. bones, muscles). RAD1901 or salt orsolvate (e.g., hydrate) thereof may be especially useful in treatingmetastatic cancers and/or tumors having overexpression of ER in otherorgans, e.g., the original breast cancer, uterus cancer, and/or ovarycancer migrated to other organs (e.g., bones, muscles), to treat breastcancer, uterus cancer, and/or ovary cancer lesions in other organs(e.g., bones, muscles), without negative effect to said organs.

III(B). RAD1901 Decreased ER Expression and Inhibited ER Pathway.

III(B)(i)(1) RAD1901-Palbo Combo was More Effective in Decreasing ER andPR Expression in MCF7 Xenograft Models and Treatment with RAD1901,Palbociclib or Fulvestrant Alone, or a Ful-Palbo Combination.

Treatment with the combination of RAD1901 and palbociclib was also moreeffective at decreasing ER and PR expression in vivo in the MCF7xenograft models (as described in Example I(A)(ii)) than treatment withRAD1901, palbociclib, or fulvestrant alone, or treatment with acombination of fulvestrant and palbociclib (FIG. 13); tumors harvestedtwo hours after the last dosing).

III(B)(i)(2) Comparison of RAD1901 and Fulvestrant in MCF7 and T47D CellLines.

The effects of RAD1901 and fulvestrant were compared using MCF7 and T47Dcell lines, both are human breast cancer cell lines, at variousconcentrations, 0.01 μM, 0.1 μM and 1 μM (FIG. 14A for MCF7 cell lineassays; and FIG. 14B for T47D cell lines). Three ER target genes,progesterone receptor (PgR), growth regulation by estrogen in breastcancer 1 (GREB1) and trefoil factor 1 (TFF1), were used as markers.RAD1901 caused ER degradation and inhibited ER signaling (FIG. 14).Unexpectedly, RAD1901 was comparable or more effective than fulvestrantin inhibiting tumor growth, and driving tumor regression as disclosedsupra in Example I(A) and Example I(B).

III(B)(i)(3) RAD1901 Treatment Resulted in ER Degradation and Abrogationof ER Signaling in MCF7 Xenograft Model—Described Supra in ExampleI(A)(ii)(1).

RAD1901 treatment resulted in ER degradation in vivo (FIGS. 15A-C,student's t-test: *p-value<0.05, **p-value<0.01) and inhibited of ERsignaling in vivo (FIGS. 15A and 15C, student's t-test: *p-value<0.05,**p-value<0.01).

Tumor harvested from MCF7 xenograft 2 hours after the final dose ofRAD1901 (30 mg/kg, 60 mg/kg, p.o., q.d.) or fulvestrant (3 mg/dose,s.c., qwk) showed significantly decreased ER and PR expression (FIGS.15A-B). Tumor harvested from MCF7 xenograft 8 hours after the final doseof fulvestrant treatment showed varied PR and ER expression. However,tumor harvested from MCF7 xenograft 8 hours after the final dose ofRAD1901 treatment showed reduced PR and ER expression (FIGS. 15A and15C).

Tumor harvested from MCF7 xenograft 8 hours or 12 hours after the singledose of RAD1901 (30 mg/kg, 60 mg/kg, or 90 mg/kg, p.o., q.d.) showedrapidly decreased PR expression (FIG. 16A). Tumor harvested from MCF7xenograft 4 hours or 24 hours after the 7th dose of RAD1901 (30 mg/kg,60 mg/kg, or 90 mg/kg, p.o., q.d.) showed consistent and stableinhibition of ER signaling (FIG. 16B). Quantification of western blotanalyses of tumor harvested from MCF7 xenograft at various time pointsduring the treatment of RAD1901 (30 mg/kg, 60 mg/kg, or 90 mg/kg, p.o.,q.d.) showed a dose-dependent decrease in PR (FIG. 16c ).

RAD1901 treatment caused a rapid decrease in proliferation in MCF7xenograft models. For example, tumor harvested from MCF7 xenograftmodels 8 hours after the single dose of RAD1901 (90 mg/kg, p.o., q.d.)and 24 hours after the 4th dose of RAD1901 (90 mg/kg, p.o., q.d.) weresectioned and stained to show a rapid decrease of the proliferationmarker Ki67 (FIGS. 17A-B).

These results suggest that RAD1901 treatment results in ER degradationand inhibition of ER signaling in ER WT xenografts in vivo.

III(B)(i)(4) RAD1901 Treatment Resulted in ER Degradation and Abrogationof ER Signaling in PDx-4 Models Described Supra in Example I(A)(ii).

RAD1901 treatment caused a rapid decrease in proliferation in the PDx-4models. For example, four hours after the final dose on the last day ofa 56 day efficacy study, tumor harvested from PDx-4 models treated withRAD1901 (30, 60, or 120 mg/kg, p.o., q.d.) or fulvestrant (1 mg/animal,qwk) were sectioned and showed a rapid decrease of the proliferationmarker Ki67 compared to PDx-4 models treated with fulvestrant (FIG. 18).

These results suggest that RAD1901 treatment results in ER degradationand inhibition of ER signaling in ER WT xenografts in vivo.

III(B)(ii) RAD1901 Treatment Resulted in Decreased ER Signaling in aMutant ER Xenograft PDx-5 Models Described Supra in ExampleI(A)(iii)(1).

Tumors were harvested at the indicated time points after the last day ofdosing (unless otherwise specified), homogenized in RIPA buffer withprotease and phosphatase inhibitors using a Tissuelyser (Qiagen). Equalamounts of protein were separated by MW, transferred to nitrocellulosemembranes and blotted with the following antibody as described in theMaterials and methods section: progesterone receptor (PR, Cell SignalingTechnologies; 3153).

Bands were quantified using 1D Quant software (GE), and PR IHC Allredscores obtained from PDx-5 models as described in Example I(A)(iii)(1)are shown in FIG. 19. Fulvestrant exerted little influence to PRexpression, while RAD1901 showed efficacy at dosages of both 60 mg/kgand 120 mg/kg (p.o., q.d., III(B)(ii)—FIG. 1).

These results indicate that for tumors expressing certain ERα mutations(e.g., Y537S), RAD1901 was more effective than fulvestrant at inhibitingthe tumor growth, especially effective in inhibiting the growth oftumors which were hardly responsive to fulvestrant treatment (e.g., at adosage of 3 mg/dose, s.c., qwk, FIG. 6A for PDx-5). Furthermore, for thetumors which did not respond well to fulvestrant treatment (e.g.,PDx-5), RAD1901 was effective in reducing PR expression in vivo, whilefulvestrant was not (FIG. 19).

Example IV Impact of RAD1901 Treatment to Uterine Tissue and/or BMD

IV(a(1)): RAD1901 Antagonized Estradiol Stimulation of Uterine Tissue.

The uterotropic effects of RAD1901 were investigated by assessingchanges in uterine weight, histology, and C3 gene expression in immaturerats. Results from a representative study are shown in FIGS. 20A-D.

Assessment of Uterotropic Activity

Sprague-Dawley rat pups were weaned at 19 days of age, randomized intogroups (n=4), and administered vehicle (aqueous methylcellulose), E2(0.01 mg/kg), raloxifene (3 mg/kg), tamoxifen (1 mg/kg), RAD1901 alone(0.3 to 100 mg/kg), or RAD1901 (0.01 to 10 mg/kg) in combination with E2(0.01 mg/kg), either s.c. or p.o. as appropriate (see reagents, above),q.d., for 3 consecutive days. Twenty-four hours after the final dose,all animals were euthanized by carbon dioxide inhalation. Body weightsand wet uterine weights were recorded for each animal. Similar assayswere also conducted with RAD1901 (0.03 to 100 mg/kg) in rats and mice(Charles River Laboratories, Montreal, QC).

Fresh uterine tissue from each rat was fixed in 4% paraformaldehyde,dehydrated with ethanol, and embedded into JB4 plastic resin. Sectionswere cut at 8 μm and stained with 0.1% Toluidine Blue O. Thickness ofthe endometrial epithelium was measured using a Zeiss Axioskop 40microscope using the Spot Advanced program; the mean of 9 measurementsper specimen was calculated.

Uterine Complement Component 3 (C3) Gene Expression

To determine relative expression levels of C3 in the treated uterinetissue, RNA was extracted from the remaining tissue using the Micro toMidi Total RNA Purification Kit (Invitrogen, Carlsbad, Calif.) accordingto the manufacturer's instructions. RNA was quantified, and equalamounts were reverse-transcribed using the High Capacity cDNA ArchiveKit (Applied Biosystems, Foster City, Calif.).

Quantitative PCR was performed using the ABI Prism 7300 System (AppliedBiosystems). PCR was done using the Taqman Universal Master Mix withprobe sets for C3 and for the 18S ribosomal RNA as a reference gene.Thermal cycling conditions comprised an initial denaturation step at 95°C. for 10 min, followed by 40 cycles at 95° C. for 15 second and 60° C.for 1 minute.

Relative gene expression was determined by normalizing each sample tothe endogenous control (18S) and comparing with a calibrator (vehicle).Relative gene expression was determined using the following equation:2-ΔΔCt (where Ct=cycle threshold or the cycle number at which PCRproduct was first detected, ΔCt=normalized sample value, andΔΔCt=normalized difference between dosed subjects and the vehicle). Fivereplicate gene expression determinations were conducted for each dose,within each study.

Treatment with E2 (0.01 mg/kg), raloxifene (RAL, 3 mg/kg) or tamoxifen(TAM, 1 mg/kg) resulted in significant increases in uterine wet weightcompared to vehicle alone, whereas RAD1901 treatment at a range of dosesbetween 0.3 and 100 mg/kg did not significantly affect uterine wetweight (FIG. 20A). Data shown (FIG. 20A) are means (±SEM); n=4 rats pergroup; P vs. vehicle: *<0.05; vs. E2: ‡<0.05. Further, when administeredin combination with E2 (0.01 mg/kg), RAD1901 antagonized E2-mediateduterine stimulation in a dose-dependent manner, exhibiting significantinhibition of uterotropic activity at doses of 0.1 mg/kg and greater andcomplete inhibition at 3 mg/kg. The EC₅₀ for RAD1901 was approximately0.3 mg/kg. Similar results were obtained in mice where RAD1901 doses0.03 to 100 mg/kg also had no effect on uterine wet weight or epithelialthickness (data not shown).

Treatment-dependent changes in uterine tissue were further investigatedby quantitative microscopic histology. There was a statisticallysignificant increase in endometrial epithelial thickness after treatmentwith E2 at both 0.01 and 0.3 mg/kg (FIG. 20B). A significant increase inepithelial thickness was also observed after treatment with tamoxifen (1mg/kg) or raloxifene (3 mg/kg). In contrast, RAD1901 treatment did notincrease endometrial epithelial thickness up to the highest evaluateddose of 100 mg/kg. Representative images of the endometrial epitheliumare shown in FIG. 20C.

Consistent with the changes in both uterine weight and endometrialepithelial thickness, E2, tamoxifen, and raloxifene all significantlyincreased the expression of the estrogen-regulated complement gene, C3(FIG. 20D)). In contrast, RAD1901 did not increase C3 gene expression atany of the doses tested (0.3 to 100 mg/kg). Furthermore, RAD1901 at 1, 3and 10 mg/kg significantly suppressed E2-stimulated C3 gene expression.

RAD1901 Did not Stimulate the Uterus of Immature Female Rats

Immature female rats were administered p.o., q.d., for 3 consecutivedays with vehicle (VEH), estradiol (E2), Raloxifene (RAL), Tamoxifen(TAM), RAD1901 or RAD1901+E2. Wet uterine weights were measured. Datashown (FIG. 20A) are means (±SEM); n=4 rats per group; P vs. vehicle:*<0.05; vs. E2: ‡<0.05.

Example II(A)(2). Treatment with RAD1901 Protected Against Bone Loss inOvariectomized Rats

The bone-specific effects of RAD1901 was examined in ovariectomizedrats.

As a model of postmenopausal bone loss, ovariectomy was performed onanesthetized adult female Sprague-Dawley rats, with sham surgery as acontrol. Following surgery, ovariectomized rats were treated q.d. for 4weeks with vehicle, E2 (0.01 mg/kg), or RAD1901 (0.1, 0.3, 1, 3 mg/kg),administered as described above, with 20 animals per group. Animals inthe sham surgery group were vehicle treated. All animals were euthanizedby carbon dioxide inhalation 24 hours after the final dose. Bone mineraldensity was assessed at baseline and again after 4 weeks of treatmentusing PIXImus dual emission x-ray absorptiometry.

At necropsy, the left femur of each animal was removed, dissected freeof soft tissue and stored in 70% ethanol before analysis. A detailedqualitative and quantitative 3-D evaluation was performed using amicro-CT40 system (Scanco Systems, Wayne, Pa.). For each specimen, 250image slices of the distal femur metaphysis were acquired. Morphometricparameters were determined using a direct 3-D approach in pre-selectedanalysis regions. Parameters determined in the trabecular bone includedbone volume density, bone surface density, trabecular number, trabecularthickness, trabecular spacing, connectivity density, and apparent bonedensity.

Following ovariectomy, untreated (vehicle control) rats experienced adecrease in bone mineral density both in the whole full femur and in thelumbar spine compared to baseline (Table 5). Treatment with E2 wasassociated with prevention of bone loss in both the femur and spine.Treatment with RAD1901 resulted in a dose-dependent and statisticallysignificant suppression of ovariectomy-induced bone loss (data shown forthe 3 mg/kg treatment group). At doses of 0.1 mg/kg to 3 mg/kg, bonemineral density in RAD1901-treated rats was complete, with nostatistically significant difference from the E2-treated group.

Micro-CT analysis of the distal femur (Table 6) demonstrated thatovariectomy induced significant changes in a number of keymicro-architectural parameters when compared to sham surgery animals.These changes were consistent with a decrease in bone mass and includedecreased bone volume, reduced trabecular number, thickness and density,and increased trabecular separation. Consistent with the preservation ofbone mineral density observed after treatment with RAD1901, significantpreservation of trabecular architecture was observed in keymicro-structural parameters (Table 6)

Example IV(B): Phase 1 Dose Escalation Study of RAD101 in HealthyPostmenopausal Women

In the phase 1 study, safety, tolerability and pharmacokinetics wereevaluated in 44 healthy postmenopausal females. No dose limitingtoxicities (DLT) were observed, maximum tolerated dose (MTD) was notestablished. Plasma exposure increased more than dose proportionallyover the dose range tested.

Subjects

44 healthy postmenopausal females were enrolled as subjects for thisphase 1 study. The subjects had an amenorrhea duration of at least 12months and serum FSH consistent with menopause. The subjects were 40-75years old with BMI of 18.0-30 kg/m². Subjects having evidence ofclinically relevant pathology, increased risk of stroke or of historyvenous thromboembolic events, or use of concomitant medication less than14 days prior to admission to clinical research center (paracetamolallowed up to 3 days prior) were excluded.

Dosing

The subjects were treated with placebo or at least one dose p.o., q.d.after a light breakfast for 7 days at dose levels of 200 mg, 500 mg, 750mg and 1000 mg, respectively. The key baseline demographics of the 44healthy postmenopausal females enrolled in the phase 1 study aresummarized in Table 7.

Treatment Emergent Adverse Events (TEAEs)

TEAEs were recorded, and the most frequent (>10% of patients in thetotal active group who had any related TEAEs) adverse events (AEs) aresummarized in Table 8, “n” is number of subjects with at least onetreatment-related AE in a given category, AEs graded as per the CommonTerminology Criteria for Adverse Events (CTCAE) v4.0, and any patientwith multiple scenarios of a same preferred term was counted only onceto the most severe grade. No dose limiting toxicities were observed,maximum tolerated dose (MTD) was not established.

Pharmacokinetic Evaluations

A series of blood samples were taken during the study for the analysisof RAD1901 in plasma. Blood samples of 5 mL each were taken via anindwelling IV catheter or by direct venipuncture into tubes containingK₃-EDTA as anticoagulant. Steady state was achieved by day 5 oftreatment. Geometric Mean (Geo-Mean) plasma concentration-time profilesof RAD1901 were evaluated. Plasma pharmacokinetic results of the groupstreated with RAD1901 (200, 500, 750 or 1,000 mg) on Day 7 (N=35) in thestudy are provided in Table 8 and FIG. 21, as an example. The mediant_(1/2) was between 37.5-42.3 hours (Table 8). After multiple doseadministration of RAD1901, median t_(max) ranged between 3-4 hourspost-dose.

Example V(A)-1. Modeling of RAD1901-ERα Binding Using Select PublishedER Structures

Unless specified otherwise, when structures are shown by their stickmodel, each end of a bond is colored with the same color as the atom towhich it is attached, wherein grey is carbon, red is oxygen, blue isnitrogen and white is hydrogen.

Fourteen published structures (i.e., models) of ERα ligand-bindingdomain (LBD) complexed with various ER ligands were selected from 96published models by careful evaluation. One of these fourteen models was3ERT (human ERα LBD bound to 4-hydroxytamoxifen (OHT)). OHT is theactive metabolite of tamoxifen and a first generation SERM thatfunctions as an antagonist in breast tissue.

In 3ERT (FIGS. 21 and 22), the ERα binding site adopts a three layer“helical sandwich” forming a hydrophobic pocket which includes Helix 3(H3), Helix 5 (H5), and Helix 11 (H11) (FIG. 21). The dotted box in FIG.22 represents the binding site and residues within the binding site thatare important or are effected by OHT binding. OHT functions as anantagonist by displacing H12 into the site where LXXLL coactivator(s)binds. OHT occupies the space normally filled by L540 and modifies theconformation of four residues on the C-terminal of Helix 11 (G521, H524,L525, and M528). OHT also forms a salt bridge with D351, resulting incharge neutralization.

The other thirteen ERα LBD-ER ligand models were compared to 3ERT.Differences in their residue poses are summarized in Table 10.Superimposition of the ERα structures of the fourteen models (FIG. 23)shows that these structures differed significantly at residues E380,M421, G521, M522, H524, Y526, 5527, M528, P535, Y537, L540, and variouscombinations thereof.

Root-mean-square deviation (RMSD) calculations of any pair of thefourteen models are summarized in Table 11. Structures were consideredto be overlapping when their RMSD was <2 Å. Table 11 shows that allfourteen models had a RMSD<1.5 Å. Using conditional formatting analysissuggested that 1R5K and 3UUC were the least similar to the other models(analysis not shown). Therefore, 1R5K and 3UUC were considered a unique,separate structural cluster to be examined.

ERα residues bound by ligand in the fourteen models are summarized inTable 12. Table 12 also shows the EC₅₀ in the ERα LBD-antagonistcomplexes. Out of the fourteen models, thirteen showed H-bondinteractions between the ligand and E353; twelve showed pi interactionsbetween the ligand and F404; five showed H-bond interactions between theligand and D351; six showed H-bond interactions between the ligand andH524; four showed H-bond interactions between the ligand and R394; andone (3UUC) showed interactions between the ligand and T347.

Each of the fourteen models was used to dock a random library of 1,000compounds plus the ligand the model was published with (the knownantagonist) to determine whether the model could identify and prioritizethe known antagonist. If the model could identify the known antagonist,the model was determined to be able to predict the pose of its ownpublished ligand. EF₅₀ was then calculated to quantify the model'sstrength to see how much better it was than a random selection. RAD1901was docked in the selected models (e.g., FIGS. 24-28). Docking scores ofthe published ligand and RAD1901 in the models were determined. EC₅₀ wasalso determined. Visual inspection of RAD1901 showed that it “obeyed”the interactions shown with the published ligands in 1R5K, 1SJ0, 2JFA,2BJ4, and 2OUZ. No spatial clashes were noticed. In certain embodiments,e.g., in 1R5k and 2BJ4, RAD1901 had a higher docking score than thepublished ligand.

The evaluation results of nine models (1ERR, 3ERT, 3UCC, 210K, 1R5K,1SJ0, 2JFA, 2BJ4, and 2OUZ) are summarized in Table 13.

1ERR and 3ERT could not predict the correct pose of their crystallizedligand. RAD1901 did not dock in 3UCC. The tetrahydronaphtaalen in210K-RAD1901 bound in a non-traditional manner.

The major differences between the models 1R5K, 1SJ0, 2JFA, 2BJ4, and2OUZ were the residues in the C-term of Helix 11 (G521-M528).

FIG. 24 shows the modeling of RAD1901-1R5K (a) and GW5-1R5K (b). RAD1901bound with H-bond interactions to E353, R394, and L536; and withp-interaction with F404.

FIG. 25 shows the modeling of RAD1901-1SJ0 (a) and E4D-1SJ0 (b). RAD1901bound with H-bond interactions to E353, and D351; and with p-interactionwith F404.

FIG. 26 shows the modeling of RAD1901-2JFA (a) and RAL-2JFA (b). RAD1901bound with p-interaction with F404.

FIG. 27 shows the modeling of RAD1901-2BJ4 (a) and OHT-2BJ4 (b). RAD1901bound with H-bond interactions with E353 and R394; and p-interactionwith F404.

FIG. 28 shows the modeling of RAD1901-2IOK (a) and IOK-2IOK (b). RAD1901bound with H-bond interactions with E353, R394, and D351; andp-interaction with F404.

The published ligands in the models have the following structures:

Example V(A)-2. Induced Fit Docking (IFD) of ERα with RAD1901 andFulvestrant

Binding conformation of RAD1901 in ERα was further optimized by IFDanalysis of the five ERα crystal structures 1R5K, 1SJ0, 2JFA, 2BJ4, and2OUZ. IFD analysis accounted for the receptor flexibility (upon ligandbinding) to accommodate its correct binding conformation.

A library of different conformations for each ligand (e.g., RAD1901 andfulvestrant) was generated by looking for a local minima as a functionof rotations about rotatable bonds. The library for RAD1901 had 25different conformations.

The five ERα crystal structures were prepared and minimized. Thecorresponding ligand in the published X-ray structures were used todefine the ERα binding pocket.

RAD1901 conformations were docked into the prepared ERα structureswherein they were allowed to induce side-chain or back-bone movements toresidues located in the binding pocket. Those movements allowed ERα toalter its binding site so that it was more closely conformed to theshape and binding mode of the RAD1901 conformation. In some examples,small backbone relaxations in the receptor structure and significantside-chain conformation changes were allowed in the IFD analysis.

An empirical scoring function was used to approximate the ligand bindingfree energy to provide a docking score or Gscore. Gscore is also knownas GlideScore, which may be used interchangeably with docking score inthis example. The docking score was an estimate of the binding affinity.Therefore, the lower the value of the docking score, the “better” aligand bound to its receptor. A docking score of −13 to −14 correspondedto a very good binding interaction.

The RAD1901 conformations resulted from the IFD analysis with 1R5K,1SJ0, 2JFA, 2BJ4, and 2OUZ respectively were superimposed to show theirdifferences (FIG. 29-31, shown in stick model). All bonds in eachRAD1901 conformation were shown in the same color in FIGS. 29, 30 and31(a).

The RAD1901 conformations resulted from the IFD analysis with 1R5K(blue) and 2OUZ (yellow) had N-benzyl-N-ethylaniline group of RAD1901 onthe front (FIG. 29). The RAD1901 conformations resulted from the IFDanalysis with 2BJ4 (green) and 2JFA (pink) had N-benzyl-N-ethylanilinegroup of RAD1901 on the back (FIG. 30). The RAD1901 conformationsresulted from the IFD analysis with 2BJ4 (green), 2JFA (pink) and 1SJ0(brown) were quite similar as shown by their superimpositions (FIGS.31(a) and (b)). The RAD1901 IFD docking scores are summarized inV(A)—Table 5.

The IFD of RAD1901 with 2BJ4 showed hydrogen bond interactions with E353and D351 and pi-interactions with F404 (FIG. 32(a)-(c)). FIG. 32(a)showed regions within the binding site suitable for H-bond acceptorgroup (red), H-bond donor group (blue) and hydrophobic group (yellow).In FIG. 32(a)-(b), light blue was for carbon for RAD1901. FIG. 33(a)-(c)show a protein-surface interactions of the IFD of RAD1901 with 2BJ4.V(A)—FIGS. 33(a) and (b) are the front view, and FIG. 33(c) is the sideview. The molecular surface of RAD1901 was blue in FIG. 33(a), and greenin FIG. 33(c). FIG. 33(b)-(c) are electrostatic representation of thesolvent accessible surface of ERα, wherein red representedelectronegative and blue represented electropositive.

Similar IFD analysis was carried out for fulvestrant with 2BJ4 asdescribed supra. The fulvestrant-2BJ4 IFD resulted in a Gscore of−14.945 and showed hydrogen bond interactions with E353, Y526, and H524and pi-interactions with F404 (FIG. 34(a)-(c)). FIG. 34(a) showedregions within the binding site suitable for H-bond acceptor group(red), H-bond donor group (blue) and hydrophobic group (yellow). In FIG.34(a), light blue was for carbon for RAD1901.

FIG. 35(a)-(b) showed RAD1901 and fulvestrant docked in 2BJ4 by IFD bothhad pi-interactions with F404 and hydrogen bond interactions with E353.Furthermore, RAD1901 had hydrogen bond interaction with D351 (bluerepresenting RAD1901 molecular surface, FIG. 35(b)), while fulvestranthad hydrogen bond interactions with Y526, and H524 (green representingfulvestrant molecular surface, FIG. 35(c)). Superimpositions of 2BJ4docked with RAD1901 and fulvestrant are shown in FIG. 36(a)-(b). In FIG.36(a), green represents fulvestrant molecular surface and bluerepresents RAD1901 molecular surface. In FIG. 36(b), the brown structureis fulvestrant and the blue structure is RAD1901.

Example V(A)-3. Modeling Evaluation of Select ERα Mutations

Effects of various ERα mutations on the C-terminal ligand-binding domainwere evaluated. Specific ERα mutations evaluated were Y537X mutantwherein X was S, N, or C; D538G; and S463P.

Y537 resides in Helix 12. It may regulate ligand binding,homodimerization, and DNA binding once it is phosphorylated, and mayallow ERα to escape phosphorylation-mediated controls and provide a cellwith a potential selective tumorigenic advantage. In addition, it maycause conformational changes that makes the receptor constitutivelyactive.

The Y537S mutation favors the transcriptionally active closed pocketconformation, whether occupied by ligand or not. The closed butunoccupied pocket may account for ERα's constitutive activity (Carlsonet al. Biochemistry 36:14897-14905 (1997)). Ser537 establishes ahydrogen-bonding interaction with Asp351 resulting in an alteredconformation of the helix 11-12 loop and burial of Leu536 in asolvent-inaccessible position. This may contribute to constitutiveactivity of the Y537S mutant protein. The Y537S surface mutation has noimpact on the structure of the LBD pocket.

Y537N is common in ERα-negative metastatic breast cancer. A mutation atthis site may allow ERα to escape phosphorylation-mediated controls andprovide a cell with a potential selective tumorigenic advantage.Specifically, Y537N substitution induces conformational changes in theERα that might mimic hormone binding, not affecting the ability of thereceptor to dimerize, but conferring a constitutive transactivationfunction to the receptor (Zhang et al. Cancer Res 57:1244-1249 (1997)).

Y537C has a similar effect to Y537N.

D538G may shift the entire energy landscape by stabilizing both theactive and inactive conformations, although more preferably the active.This may lead to constitutive activity of this mutant in the absence ofhormones as observed in hormone-resistant breast cancer (Huang et al.,“A newfound cancer-activating mutation reshapes the energy landscape ofestrogen-binding domain,” J. Chem. Theory Comput. 10:2897-2900 (2014)).

None of these mutations are expected to impact the ligand binding domainnor specifically hinder RAD1901 binding. Y537 and D538 may causeconformational changes that leads to constitutive receptor activationindependent of ligand binding.

Example V(B). In Vitro Binding Assay of ERα Constructs of Wildtype andLBD Mutant with RAD1901 and Other Compounds

In vitro binding assay of ERα constructs of wildtype (WT) and LBD mutantwith RAD1901 showed that RAD1901 bound to mutant ERα with a similaraffinity as to WT ERα.

ERα constructs of WT and LBD mutant were prepared by expressing andpurifying the corresponding LBD residues 302-552 with N-terminalthioredoxin and 6×His tags which were cleaved by TEV protease.

Fluorescence polarization (FP) was used to determine binding of testcompounds (RAD1901, fulvestrant, bazedoxifene, raloxifene, tamoxifene,and AZD9496) to ERα as per manufacturer's instructions (Polar Screen,Invitrogen) with 2 nM fluoromone, 100 nM ERα construct of WT or LBDmutant. Each set was carried out in duplicate and tested one testcompound to determine the IC₅₀ for different ERα constructs (FIG. 38 forRAD1901 binding essay).

As stated above, the foregoing is merely intended to illustrate variousembodiments of the present invention. The specific modificationsdiscussed above are not to be construed as limitations on the scope ofthe invention. It will be apparent to one skilled in the art thatvarious equivalents, changes, and modifications may be made withoutdeparting from the scope of the invention, and it is understood thatsuch equivalent embodiments are to be included herein. All referencescited herein are incorporated by reference as if fully set forth herein.

TABLE 1 RAD1901 levels in plasma, tumor and brain of mice implanted withMCF7 cells after treated for 40 days. Dose Plasma Tumor Brain B/P T/P(mg/kg) (ng/mL) (ng/mL) (ng/mL) Ratio Ratio Vehicle BLQ* BLQ BLQ — —RAD1901 0.3 2 11 BLQ — RAD1901 1 3 45 BLQ — RAD1901 3 9 169 7 0.78 18.78RAD1901 10 39 757 14 0.36 19.41 RAD1901 30 137 3875 72 0.53 28.28RAD1901 60 334 11117 201 0.60 33.28 *BLQ: below the limit ofquantitation

TABLE 2 SUV for uterus, muscle, and bone for a human subject treatedwith 200 mg dose p.o., q.d., for six days Uterus SUV Bone SUV Muscle SUVDose % Change % Change % Change 200 mg −85% 16% 0%

TABLE 3 SUV for uterus, muscle, and bone for human subjects (n = 4)treated with 500 mg dose p.o., q.d., for six days. Uterus Mean SUV MeanUterus Change Muscle Muscle SUV Mean Bone Bone SUV Subject # Scan SUV(%) SUV Change (%) SUV Change (%) 1 Baseline 3.88 0.33 0.36 Day 6 0.58−85 0.31 −6 0.48 33 2 Baseline 6.47 0.25 0.49 Day 6 0.33 −86 0.42 680.55 12 3 Baseline 3.66 0.50 0.41 Day 6 0.58 −84 0.31 −38 0.47 −23 4Baseline 3.35 0.30 0.40 Day 6 0.41 −88 0.24 −20 0.52 30 Mean −86 1 13

TABLE 4 Effect of RAD1901 on BMD in ovariectomized rats.^(a) Femur BMDLumbar Spine BMD Treatment (% change) (% change) Sham  3.1 ± 2.4*  2.7 ±5.0* OVX + veh −5.4 ± 5.1  −10.2 ± 12.8 OVX + E2 −0.5 ± 2.6*  −2.1 ±12.2* OVX + RAD1901  0.4 ± 2.8*  −1.1 ± 79* ^(a)Adult female ratsunderwent either sham or ovariectomy surgery before treatment initiationwith vehicle, E2 (0.01 mg/kg) or RAD1901 (3 mg/kg) q.d. (n = 20 pertreatment group). BMD was measured by dual emission x-ray absorptiometryat baseline and after 4 weeks of treatment. Data are expressed as mean ±SD. *P < 0.05 versus the corresponding OVX + Veh control. BMD, bonemineral density; E2, beta estradiol; OVX, ovariectomized; Veh, vehicle.

TABLE 5 Effect of RAD1901 on femur microarchitecture in ovariectomizedrats^(a) BV/TV ConnD TbN TbTh TbSp ABD Treatment (%) (1/mm³) (1/mm) (mm)(mm) (mgHA/ccm) Sham 0.394 ± 0.069* 138 ± 21* 5.2 ± 0.6* 0.095 ± 0.008*0.175 ± 0.029* 456 ± 61* OVX + Veh 0.234 ± 0.065  91 ± 32 3.5 ± 0.90.085 ± 0.011 0.307 ± 0.086 301 ± 69 OVX + E2 0.309 ± 0.079* 125 ± 25*4.8 ± 0.8* 0.086 ± 0.008 0.204 ± 0.054* 379 ± 75* OVX + RAD1901 0.300 ±0.066* 113 ± 22* 4.5 ± 0.8* 0.088 ± 0.008 0.218 ± 0.057* 370 ± 66*^(a)Adult female rats underwent either sham or ovariectomy surgerybefore treatment initiation with vehicle, E2 (0.01 mg/kg) or RAD1901 (3mg/kg) q.d. (n = 20 per treatment group). After 4 weeks, Bonemicroarchitecture was evaluated using microcomputed tomography. Data areexpressed as mean ± SD. *P < 0.05 versus the corresponding OVX + Vehcontrol. ABD, apparent bone density; BV/TV, bone volume density; ConnD,connectivity density; E2, beta estradiol; OVX, ovariectomized; TbN,trabecular number; TbTh, trabecular thickness; TbSp, trabecular spacing;Veh, vehicle.

TABLE 6 Key baseline demographics of Phase 1 dose escalation study ofRAD1901 RAD1901 RAD1901 RAD1901 RAD1901 Placebo 200 mg 500 mg 750 mg1,000 mg (N = 8) (N = 15) (N = 14) (N = 8) (N = 7) Race white 8(100)14(93) 10(71) 8(100) 7(100) (% of the cohort) Mean age, 64 62 59 64 64years Mean BMI, 26.1 25 24.4 24.9 26.7 kg/m²

TABLE 7 Most frequent (>10%) treatment related AEs in a Phase 1 doseescalation study of RAD1901 Placebo 200 mg 500 mg 750 mg N = 8 N = 15 N= 14 N = 8 n (%) n (%) n (%) n (%) Gr1 Gr2 Gr3 Gr1 Gr2 Gr3 Gr1 Gr2 Gr3Gr1 Gr2 Gr3 Nausea 2 (25) 0 0 5 (33) 0 0 3 (21) 2 (14) 0 2 (25) 1 (13) 0Dyspepsia 1 (13) 0 0 3 (20) 0 0 5 (36) 2 (14) 0 4 (50) 0 0 Vomiting 0 00 2 (13) 0 0 1 (7)  5 (36) 1 (7) 0 2 (25) 0 Hot flush 1 (13) 0 0 2 (13)0 0 6 (43) 0 0 2 (25) 0 0 Abdominal pain 1 (13) 0 0 2 (13) 2 (13) 0 3(21) 0 0 1 (13) 0 0 Oesophageal pain 0 0 0 0 2 (13) 0 1 (7)  3 (21) 0 1(13) 0 0 Headache 0 0 0 3 (20) 0 0 1 (7)  1 (7)  0 3 (38) 0 0 Hiccups 00 0 1 (7) 0 0 4 (29) 0 0 2 (25) 0 0 Salivary hypersecretion 0 0 0 2 (13)0 0 2 (14) 0 0 2 (25) 0 0 Diarrhoea 1 (13) 0 0 0 0 0 3 (21) 0 0 0 0 0Dysphagia 0 0 0 0 0 0 1 (7)  2 (14) 0 3 (38) 1 (13) 0 Sensation of aforeign body 0 0 0 2 (13) 0 0 1 (7)  0 0 0 0 0 Abdominal distension 0 00 1 (7)  1 (7)  0 1 (7)  0 0 1 (13) 0 0 Odynophagia 0 0 0 2 (13) 0 0 1(7)  1 (7)  0 0 0 0 Dizziness 2 (25) 0 0 1 (7)  D 0 2 (14) 0 0 1 (13) 00 Abdominal discomfort 0 0 0 3 (20) 0 0 0 0 0 1 (13) 1 (13) 0 Flatulence0 0 0 2 (13) 0 0 2 (14) 0 0 1 (13) 0 0 Myalgia 1 (13) 0 0 2 (13) 1 (7) 0 0 1 (7)  0 1 (13) 0 0 1000 mg Total Active Total N = 7 N = 44 TEAE n(%) n (%) N = 44 Gr1 Gr2 Gr3 Gr1 Gr2 Gr3 All Nausea 4 (57) 2 (29) 0 14(32)  5 (11) 0 19 (43) Dyspepsia 1 (14) 1 (14) 0 13 (30) 3 (7) 0 16 (36)Vomiting 0 3 (43) 0 3 (7) 10 (23) 1 (2) 14 (32) Hot flush 1 (14) 0 0 11(25) 0 0 11 (25) Abdominal pain 1 (14) 1 (14) 0  7 (16) 3 (7) 0 10 (23)Oesophageal pain 1 (14) 1 (14) 1 (14) 3 (7)  6 (14) 1 (2) 10 (23)Headache 2 (29) 0 0  9 (20) 1 (2) 0 10 (23) Hiccups 2 (29) 0 0  9 (20) 00  9 (20) Salivary hypersecretion 2 (29) 0 0  8 (18) 0 0  8 (18)Diarrhoea 3 (43) 1 (14) 0  6 (14) 1 (2) 0  7 (16) Dysphagia 0 0 0 4 (9)3 (7) 0  7 (16) Sensation of a foreign body 4 (57) 0 0  7 (16) 0 0  7(16) Abdominal distension 2 (29) 0 0  5 (11) 1 (2) 0  6 (14) Odynophagia1 (14) 1 (14) 0 4 (9) 2 (5) 0  6 (14) Dizziness 1 (14) 0 0  5 (11) 0 0 5 (11) Abdominal discomfort 0 0 0 4 (9) 1 (2) 0  5 (11) Flatulence 0 00  5 (11) 0 0  5 (11) Myalgia 0 0 0 3 (7) 2 (5) 0  5 (11)

TABLE 8 Pharmacokinetic parameters in a Phase 1 dose escalation study ofRAD1901 (Day 7) 200 mg 500 mg 750 mg 1000 mg Parameter Statistic N = 15N = 11 N = 6 N = 3 C_(max) Geo-Mean 49.8 197 322 540 (ng/mL) Min, Max30.6, 85.5 105, 316 248, 420 481, 602 t_(max) (h) Median 3.00 4.00 3.004.00 Min, Max 2.00, 6.00 2.00-6.02 3.00, 4.00 3.00, 6.00 AUC_(0-tau)Geo-Mean 670 2927 4614 8292 (h * ng/mL) Min, Max 418, 1181 1562, 54603209, 7183 7281, 8947 t_(1/2) (h) Geo-Mean 38.3 37.5 38.4 42.3 Min, Max27.7, 51.4 33.8, 41.3 34.6, 46.4 38.7, 49.4

TABLE 9 Frequency of LBD mutations Frequency (%) D538G 29.5 Y537S 25.0Y537N 13.6 Y537C 9.1 E380Q 6.8 S463P 4.5 L536R 2.3 L536Q 2.3 P535H 2.3V392I 2.3 V534E 2.3

TABLE 10 Differences of ER-α LBD-antagonist complexes in residue posesversus 3ERT Residue L1-3/ #/ Helix 8 Helix 11 Helix 5 Helix 12 PDB M421I424 E521 M524 H524 L525 Y526 S527 M528 E380 Y537 L540 2BJ4 x x x x x xx NA 2JFA x x x x x x x NA 1SJ0 x x x x x x x x 2JF9 x x x x x x x NA1YIM x x x x x x 1R5K x x x x x x X x x 1UMO x x x x 1ERR x x x x x x2IOK x x x x x x x x 3UUC x x x x x x x x 1YIN x x x X x x x x x 2AYR xX x x 2OUZ x x x x

TABLE 11 Evaluation of structure overlap of ER-α LBD-antagonistcomplexes by RMSD calculations: RMSD 3ERT 2BJ4 2JFA 1SJ0 2JF9 1Y1M 1R5K1UOM 1ERR 2IOK 3UUC 1Y1N 2AYR 3ERT 2BJ4 0.804 2JFA 1.196 0.554 1SJ00.786 0.637 1.115 2JF9 1.177 0.411 0.415 1.186 1Y1M 0.978 0.687 1.1180.276 1.072 1R5K 1.483 0.759 0.52 1.307 0.892 1.342 1UOM 0.739 0.7610.723 0.489 0.909 0.499 1.115 1ERR 1.12 0.483 0.595 1.016 0.851 1.1121.208 0.918 2IOK 0.824 0.689 0.787 0.899 0.897 0.854 1.208 0.736 0.8383UUC 1.024 0.915 0.896 1.03 0.888 1.036 1.228 1.012 0.873 0.929 1Y1N0.749 0.683 1.105 0.432 1.061 0.318 1.293 0.557 1.076 0.744 1.015 2AYR0.659 0.682 0.95 0.792 1.124 0.777 1.391 0.491 1.118 0.071 1.031 0.581

TABLE 12 Analysis of ligand binding in ER-α LBD-antagonist complexesLigand: Binding to EC₅₀ (μM) Comments 3ERT OHT: E353, R394 0.010 Flippedamine, F404 was too far from the phenol thus there were nopi-interactions 2BJ4 OHT: E353, R394, pi 0.010 F404 2JF9 OHT: E353,D351, 0.010 H524, pi F404 2JFA RAL: E353, D351, 0.002 H524 and pi F404x2 1ERR RAL: E353, D351, 0.002 Phenol flipped R394 and pi F404 x2 forH524 1YIM CM3: E353, H524, 0.0015(IC₅₀) D351-carboxyle D351 pi F404oriented well with pyrrolidine 1YIN CM3: E535, H524 pi 0.001 F404 1SJ0E4D: E353, H524, pi 0.0008(IC₅₀) F404 x 2 1R5K GW5: D351 pi F4040.039(IC₅₀) No anchor bond with E353 1UOM PTI: E353, H524 pi NA F4042IOK IOK: E353 pi F404 0.001 3UUC OD1: E353, R394, T347 NA Very smallcompound 2OUZ C3D: E353, pi F404 0.003 2AYR L4G: E353, pi F404 x2 0.0107

TABLE 13 Model evaluation for RAD1901 docking EF₅₀ Can model LigandRAD1901 EC₅₀ (=predictive predict crystal docking docking (μM) power)structure? score score 1ERR 0.001 No −11.452 −7.912 3ERT 0.002 No−12.175 −8.151 3UCC NA 8474 Yes −9.278 NA 2IOK 0.001 Yes −11.952 −10.4781R5K 0.039 6100 Yes −11.518 −12.102 1SJ0 0.001 7511 Yes −12.507 −9.8162JFA 0.001 6780 Yes −11.480 −11.055 2BJ4 0.002 5642 Yes −9.727 −11.9712OUZ 0.003 — Yes −11.789 −9.611

TABLE 14 Induced Fit Docking Score of RAD1901 with 1R5K, 1SJ0, 2IFA,2BJ4 and 2OUZ ER-α Crystal Structure RAD1901 IFD Docking Score 1R5K−14.1 1SJ0 −13.1 2JFA −13.9 2BJ4 −13.8 2OUZ −13.4

What is claimed is:
 1. A method of treating breast cancer in a subjecthaving a drug-resistant mutant estrogen receptor alpha-positive cancercomprising administering to said subject a therapeutically effectiveamount of a combination of palbociclib and RAD1901 having the structure:

or a salt or solvate thereof.
 2. The method of claim 1 wherein said drugresistant breast cancer is resistant to one or more antiestrogen oraromatase inhibitor therapies.
 3. The method of claim 2 wherein said oneor more antiestrogens are selected from the group consisting oftamoxifen, toremifene and fulvestrant and said one or more aromataseinhibitors are selected from the group consisting of aromasin, letrozoleand anastrozole.
 4. The method according to any one of claims 1-3wherein said subject expresses at least one mutant estrogen receptoralpha selected from the group consisting of D538G, Y537S, Y537N, Y537C,E380Q, S463P, L536R, L536Q, P535H, V392I and V534E.
 5. The method ofclaim 4 wherein said mutant estrogen receptor alpha is selected from thegroup consisting of Y537S, Y537N, Y537C, D538G, L536R, S463P and E380Q.6. The method according to claim 5 wherein said mutant receptor alpha isY537S.
 7. The method according to claim 1 wherein said RAD1901 isadministered in a total daily dosage of from between 100 mg and 1,500mg.
 8. The method according to claim 7 wherein said RAD1901 isadministered in a total daily dosage of from between 100 mg and 1,000mg.
 9. The method according to claim 8 wherein said RAD1901 isadministered in a total daily dosage of 100 mg, 200 mg, 300 mg, 400 mg,500 mg, 600 mg, 700 mg, 800 mg, 900 mg or 1,000 mg.
 10. The methodaccording to claim 7 wherein said daily dosage is delivered in twoseparate doses.
 11. The method according to claim 10 wherein saidseparate doses are equal doses.
 12. The method according to claim 11wherein said equal doses are 100 mg, 200 mg, 250 mg, 300 mg, 400 mg or500 mg each.
 13. The method according to claim 7 wherein said dosage isdelivered by the oral route.
 14. The method according to claim 1 whereinsaid subject is a post-menopausal woman.
 15. The method according toclaim 1 wherein said subject is first identified for treatment throughmeasuring for increased expression of one or more genes selected fromABL1, AKT1, AKT2, ALK, APC, AR, ARID1A, ASXL1, ATM, AURKA, BAP, BAP1,BCL2L11, BCR, BRAF, BRCA1, BRCA2, CCND1, CCND2, CCND3, CCNE1, CDH1,CDK4, CDK6, CDK8, CDKN1A, CDKN1B, CDKN2A, CDKN2B, CEBPA, CTNNB1, DDR2,DNMT3A, E2F3, EGFR, EML4, EPHB2, ERBB2, ERBB3, ESR1, EWSR1, FBXW7, FGF4,FGFR1, FGFR2, FGFR3, FLT3, FRS2, HIF1A, HRAS, IDH1, IDH2, IGF1R, JAK2,KDM6A, KDR, KIF5B, KIT, KRAS, LRP1B, MAP2K1, MAP2K4, MCL1, MDM2, MDM4,MET, MGMT, MLL, MPL, MSH6, MTOR, MYC, NF1, NF2, NKX2-1, NOTCH1, NPM,NRAS, PDGFRA, PIK3CA, PIK3R1, PML, PTEN, PTPRD, RARA, RB1, RET, RICTOR,ROS1, RPTOR, RUNX1, SMAD4, SMARCA4, SOX2, STK11, TET2, TP53, TSC1, TSC2,and VHL.
 16. The method according to claim 15 wherein said one or moregenes is selected from AKT1, AKT2, BRAF, CDK4, CDK6, PIK3CA, PIK3R1 andMTOR.
 17. The method according to claim 1 wherein said palbociclib isdosed at a daily dose of from between 25 mg and 250 mg.
 18. The methodaccording to claim 17 wherein said palbociclib is dosed at a daily doseof from between 50 to 125 mg.
 19. The method according to claim 17wherein said palbociclib is dosed at a daily dose of from between 75 mgto 125 mg.
 20. The method according to claim 17 wherein said palbociclibis dosed at 75 mg, 100 mg or 125 mg daily.
 21. The method according toclaim 17 wherein said palbociclib is dosed at a daily dose of frombetween 31.25 mg to 93.75 mg.
 22. The method according to claim 1wherein said palbociclib is dosed for 21 days in a 28 day cycle.
 23. Themethod according to claim 1 wherein said palbociclib is dosed less than21 days in a 28 day cycle.
 24. The method according to claim 1 whereinsaid palbociclib is dosed from between 7 days and 20 days in a 28 daycycle.
 25. The method according to claim 1 wherein said palbociclib isdosed 7 days or 14 days of a 28 day cycle.
 26. The method according toclaim 1 wherein said palbociclib is dosed in a total amount of 2,625 mgin a 28 day cycle.
 27. The method according to claim 1 wherein saidpalbociclib is dosed in a total amount of from between 656.25 mg and1,968.75 mg in a 28 day cycle.
 28. The method according to claim 1wherein said palbociclib is dosed orally.
 29. The method according toclaim 28 wherein said palbociclib is dosed orally once per day.
 30. Themethod according to claim 1, wherein the breast cancer is a metastaticbreast cancer.
 31. The method of claim 1, wherein the ratio of theconcentration of RAD1901 or a salt or solvate thereof in the tumor tothe concentration of RAD1901 or a salt or solvate thereof in plasmafollowing administration is at least about
 15. 32. The method of claim1, wherein the subject has osteoporosis or a higher risk ofosteoporosis.
 33. The method of claim 1, wherein the subject is apremenopausal woman.
 34. The method of claim 14, wherein the subject isa postmenopausal woman who had relapsed or progressed after previoustreatment with SERMs and/or AIs.
 35. The method of claim 1, wherein thesalt thereof is RAD1901 dihydrochloride.